3D cell culture encapsulation, methods and uses thereof

Abstract

A gellable material for encapsulating a cellular aggregate in a well plate is provided. The cellular aggregate, before or after encapsulation, can be organoid or a spheroid, and typically, the cellular aggregate, once encapsulated, has substantially no movement during media exchange, handling of the well plate or tilting of the well plate. Methods for producing encapsulated cellular aggregates, and uses thereof are also provided.

Description

[0001] 3D CELL CULTURE ENCAPSULATION, METHODS AND USES THEREOF

[0002] Field

[0003] The present disclosure relates to 3D cell cultures of cellular aggregates. In particular, the present disclosure relates to encapsulated cellular aggregates and methods of producing the same, for imaging and screening purposes.

[0004] Background

[0005] Organoids are three-dimensional multicellular constructs derived from primary tissue, embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) which can selforganise and self-renew and replicate at least some of the organ functionalities of the tissue from which they are derived. While 3 dimensional models are an invaluable pre-clinical tool, subsequent movement of the generated organoid or spheroid can be problematic for imaging or screening purposes.

[0006] US 20230257717 is directed to methods, compositions and kits for use in the culture of organoids in solution. A method for producing an expanded population of organoids in vitro is provided wherein the method comprises providing a population of organoid progenitor cells or organoids and culturing the population of organoids in a composition comprising a culture medium and a scaffold matrix. The scaffold matrix is present in the composition at a concentration for producing an expanded population of organoids.

[0007] A need exists for the development of a product and / or method that provides the public with a useful alternative. The background herein is included solely to explain the context of the disclosure. This is not to be taken as an admission that any of the material referred to was published, known, or part of the common general knowledge as of the priority date.

[0008] Summary

[0009] In accordance with an aspect, there is provided a gellable material for encapsulating a cellular aggregate in a well plate.

[0010] In an aspect, the cellular aggregate, once encapsulated, has substantially no movement in the well plate during media exchange, handling of the well plate or tilting of the well plate.

[0011] In an aspect, the cellular aggregate, once encapsulated, has no movement in the well plate during media exchange, handling of the well plate or tilting of the well plate. In an aspect, the cellular aggregate, once encapsulated, stays in a position in the well plate.

[0012] In an aspect, the position is relative to the x, y, and z axis of an imaging microscope.

[0013] In an aspect, the position is at a bottom of the well plate.

[0014] In an aspect, the gellable material is selected from collagen, fibronectin, Vitrogel™, iMatrix™, MD, or MC.

[0015] In an aspect, the gellable material described herein comprises a non-protein hydrogel.

[0016] In an aspect, the non-protein hydrogel comprises polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), polyglycolic acid (PGA), polyvinyl alcohol (PVA), hyaluronan (HA) gels, alginate gels, or fibrin gels.

[0017] In an aspect, the non-protein hydrogel comprises PEG.

[0018] In an aspect, the PEG is a modified PEG.

[0019] In an aspect, the modified PEG comprises a PEG crosslinked by a peptide.

[0020] In an aspect, the gellable material described herein comprises a protein hydrogel.

[0021] In an aspect, the protein hydrogel comprises collagen, elastin, gelatin, fibrin, fibronectin, or combination thereof.

[0022] In an aspect, the protein hydrogel comprises collagen.

[0023] In an aspect, the gellable material is degradable.

[0024] In an aspect, the gellable material has a low viscosity.

[0025] In an aspect, the viscosity is about 0.5 mPa.s to about 10 mPa.s

[0026] In an aspect, the gellable material is not viscous.

[0027] In an aspect, the gellable material has a convenient gelling time to encapsulate the cellular aggregate.

[0028] In an aspect, the gelling time is between about 1 minute to about 180 minutes.

[0029] In an aspect, the gelling time is about 10 minutes, 30 minutes, or 60 minutes.

[0030] In an aspect, the well plate is a flat bottom well plate.

[0031] In an aspect, the well plate is a rounded bottom well plate.

[0032] In an aspect, the well plate is made non-adherent with a non-adherent coating.

[0033] In an aspect, the non-adherent coating comprises a covalently bound hydrogel layer selected from Corning™ ultralow attachment range, Thermo Fisher™ Nunclon Sephera range, or polyHEMA (poly-2-hdroxylethyl methacrylate).

[0034] In an aspect, the cellular aggregate, before or after encapsulation, can mature into an organoid or spheroid. In an aspect, the cellular aggregate, before or after encapsulation, can mature into an organoid or spheroid for up to about 50 days.

[0035] In an aspect, the cellular aggregate, before or after encapsulation, is an organoid or a spheroid.

[0036] In an aspect, the organoid or spheroid is produced from cells derived from various tissues or organs, such as skin, breast, lung, liver, pancreas, kidney, blood, stomach, intestine, spleen, ovary, heart, cervix, bladder, oral cavity, esophagus, prostate, or mesentery.

[0037] In an aspect, the organoid or spheroid is derived from stem cells or tumor cells such as a primary tumor cell or a tumor cell line.

[0038] In an aspect, after encapsulation, the cellular aggregate is suitable for imaging or screening.

[0039] In accordance with another aspect, there is provided a method for producing an encapsulated cellular aggregate in a well plate, the method comprising: adding a gellable material to a cellular aggregate in the well plate; and forming the encapsulated cellular aggregate by gelling the gellable material.

[0040] In an aspect, the adding comprises diffusing the gellable material over a period of time.

[0041] In an aspect, the diffusing comprises adding the gellable material to the well plate gently so as to not damage the cellular aggregate.

[0042] In an aspect, the diffusing comprises layering the gellable material so that the gellable material can mix with a culture media around the cellular aggregate in the well plate.

[0043] In an aspect, the period of time is about 1 minute to about 180 minutes.

[0044] In an aspect, the gelling causes the encapsulated cellular aggregate to stay in a position in the well plate.

[0045] In an aspect, the method further comprises growing cells into the cellular aggregate in a culture medium prior to the adding the gellable material.

[0046] In an aspect, the gelling of the gellable material occurs for a period of time.

[0047] In an aspect, the period of time is about 1 minute to about 180 minutes

[0048] In an aspect, the gelling time is about 10 minutes, 30 minutes, or 60 minutes.

[0049] In an aspect, the method further comprises adding a dye compound to the well plate prior to the adding.

[0050] In an aspect, the method further comprises adding a dye compound to the well plate after the forming the encapsulated cellular aggregate.

[0051] In an aspect, the dye compound is selected from:

[0052]

[0053] , or a salt thereof.

[0054] In an aspect, the dye compound allows for detecting or monitoring changes in cell state (e.g., cell physiology, behavior) in response to an agent or stimuli such as changes in pH, temperature, salt concentration, contact with other cells, or treatment with a drug or drug candidate. In an aspect, the method further comprises adding a drug, such as an anti-cancer drug, to the encapsulated cellular aggregate.

[0055] In an aspect, the method further comprises growing cells, such as fibroblasts, on the encapsulated cellular aggregate.

[0056] In an aspect, the method further comprises maturing the cellular aggregate, before or after encapsulation, into spheroids over a period of time.

[0057] In an aspect, the method further comprises maturing the cellular aggregate, before or after encapsulation, into organoids over a period of time.

[0058] In an aspect, the period of time is about 50 days.

[0059] In an aspect, the cellular aggregate, before or after encapsulation, is a spheroid or an organoid.

[0060] In an aspect, the organoid or spheroid is produced from cells derived from various tissues or organs, such as skin, breast, lung, liver, pancreas, kidney, blood, stomach, intestine, spleen, ovary, heart, cervix, bladder, oral cavity, esophagus, prostate, or mesentery.

[0061] In an aspect, the organoid or spheroid is derived from stem cells or tumor cells such as a primary tumor cell or a tumor cell line.

[0062] In an aspect, the method further comprises adding a paramagnetic agent to the well plate prior to the adding.

[0063] In an aspect, the paramagnetic agent comprises a Gadolinium based salt or contrasting agent, optionally Gadolinium-diethylenetriamine penta-acetic acid (Gd-DTPA).

[0064] In an aspect, the well plate is a flat bottom well plate.

[0065] In an aspect, the flat bottom well plate comprises a non-adherent coating selected from Corning™ ultralow attachment range, Thermo Fisher™ Nunclon Sephera range, or polyHEMA (poly-2-hdroxylethyl methacrylate).

[0066] In an aspect, the well plate is a rounded bottom well plate.

[0067] In an aspect, after encapsulation, the cellular aggregate is suitable for imaging or screening.

[0068] In an aspect, the gellable material is selected from collagen, fibronectin, Vitrogel™, iMatrix™, MD, or MC.

[0069] In accordance with another aspect, there is provided a method of forming a nonadherent hydrogel cup for cellular aggregate formation, dispensing a non-adherent hydrogel to a well plate at a temperature, wherein the non-adherent hydrogel is sufficient viscous to form the non-adherent hydrogel cup having a curved surface. In an aspect, the method further comprises allowing the non-adherent hydrogel to cool.

[0070] In an aspect, the temperature is about 45°C to about 60°C.

[0071] In an aspect, the higher the viscosity of the non-adherent hydrogel, the steeper the sides of the cup formed.

[0072] In an aspect, well plate has a flat imaging bottom and round wells

[0073] In an aspect, the well plate does not have square wells.

[0074] In an aspect, a shape of the non-adherent hydrogel cup is determined by the viscosity of the non-adherent hydrogel while in liquid form.

[0075] In an aspect, the non-adherent hydrogel cup can support growth / differentiation of cells into the cellular aggregate.

[0076] In an aspect, non-adherent hydrogel has a refractive index close to that of the cells of the cellular aggregate.

[0077] In an aspect, non-adherent hydrogel is agarose.

[0078] In an aspect, the cellular aggregate can be encapsulated by the gellable material described herein.

[0079] In an aspect, the organoid or spheroid is produced from cells derived from various tissues or organs, such as skin, breast, lung, liver, pancreas, kidney, blood, stomach, intestine, spleen, ovary, heart, cervix, bladder, oral cavity, esophagus, prostate, or mesentery.

[0080] In an aspect, the organoid or spheroid is derived from stem cells or tumor cells such as a primary tumor cell or a tumor cell line.

[0081] In accordance with another aspect, there is provided use of the gellable material described herein for encapsulating a cellular aggregate.

[0082] In an aspect, after encapsulation, the cellular aggregate is suitable for imaging or screening.

[0083] In an aspect, the cellular aggregate, before or after encapsulation, is an organoid or a spheroid.

[0084] The novel features of the present invention will become apparent to those of skill in the art upon examination of the following detailed description of the invention. It should be understood, however, that the detailed description of the invention and the specific examples presented, while indicating certain aspects of the present invention, are provided for illustration purposes only because various changes and modifications within the spirit and scope of the invention will become apparent to those of skill in the art from the detailed description of the invention and claims that follow.

[0085] Brief Description of the Drawings

[0086] The present invention will be further understood from the following description with reference to the Figures, in which:

[0087] Figure 1 shows comparative images of organoids produced using (A) hydrogel cup or (B) flat bottom well with a non-adherent coating of polyHEMA. 5x images for 4000 MCF10A cells / well, deposited in 15 pl of media, stained with TMRE (100nM, yellow). Once cells are added to the hydrogel cups, the plate is centrifuged at 300rpm for 30 seconds, then the plate is rotated 180 degrees in the centrifuge and centrifuged for 300rpm another 30 seconds. Spheroids were formed in the flat bottom plate using the magnetic device and micrographs were recorded right after 3h incubation with 25pM paramagnetic salt prior to wash steps as washing caused the spheroids change position too much.

[0088] Figure 2 shows an image of fluorescent beads in a hydrogel cup of Figure 1 . The meniscus generated a depression of 500pm. The image demonstrates that the shape of the hydrogel cup is determined by the viscosity of the hydrogel while in liquid form. In this example, the hydrogel is low melting agarose. This cup can be used to support the growth and differentiation of the cells into organoids as well be used in the experiments wherein the spheroid / organoids are encapsulated with the gellable material as described herein.

[0089] Figure 3 shows images of the viability of cells treated with 25pM paramagnetic salt. TMRE (y axis; yellow) indicates mitochondrial inner membrane transmembrane potential and AnnV (x-axis; green) is annexin and indicates apoptotic cell death. BBSRI-5, BBSRI-7, BBSRI-8 are independent primary patient samples, incubated in conditional reprogramming medium containing the salt. The colour scale inside the area indicates cell number according to the arbitrary density scale between 0 and 1 .0 indicated below each graph. The colour scale goes from dark blue through white to dark brown. TMRE and AnnV axes are arbitrary log intensity scales from 100-10,000.

[0090] Figure 4 shows (A) images of the spheroids produced when 4000 MCF10A cells in 50 pl of media containing 25pM paramagnetic salt where indicated (+SALT) was added to each well of an imaging microtiter plate with and without a non-adherent coating (polyHEMA) and placed on the magnetic device for 3 hours. Afterwards the plate was removed from the magnetic device, incubated for 24 hours at 37 °C and 5% CO2 and then the plate was imaged by confocal microscopy with a 5X air lens. Each panel is a composite of four micrographs showing staining by Hoescht 33342 (1 pg / ml) that together provide an image of the entire well. When cells were grown for 24 hours without the salt solution in an uncoated imaging plate the cells formed a monolayer (no SALT, no coating). When incubated in the salt solution in the presence of the applied magnetic fields the cells are moved to the desired location in the well but attach to the bottom of the plate and spread out. (+ SALT, no coating). Once removed from the magnetic fields the cells will continue to spread and grow as a monolayer. When 50 pl of the cell suspension was incubated in the salt solution on a nonadherent surface (+SALT, +coating) the cells aggregated in the magnetic field and formed a spheroid. (B) images of MCF 10A spheroids after plating 4000 cells / well in 50p.L of paramagnetic salt solution in imaging wells with an anti-adherent coating (polyHEMA) dependent on the time of exposure to the paramagnetic salt. The wells were washed to remove salt prior to recording micrographs. Spheroids were generated and imaged on the same day. The images are micrographs of the MCF 10A cells recorded with a 5X air lens (4 micrographs per well as above) and stained with TMRE (1 OOnM). The yellow color shows that the spheroids are metabolically active. The washing step caused the spheroids to move and therefore they are no longer in the center of the wells. The length of time it takes for the spheroid to form determines the time at which the artificial extracellular matrix (hydrogel, not used in this experiment) and growth medium can be added to the cultures. This step dilutes the paramagnetic salt and permits medium exchange to remove the salt.

[0091] Figure 5A and B show organoids generated from 4000 MCF10A cells per well added in the indicated volumes of cell suspension on non-adherent coating on a flat bottom well of a 384 well imaging plate. (A) The cells were stained with TMRE (100nM, yellow), Annexin V- FITC (350ng / mL, green), Hoechst 33342 (1 ug / ml, blue). The spheroids were located in the wells by scanning at 5x using PreciScan software and then reimaged by confocal microscopy using a 20x air lens on the Opera Phenix automated microscope. The image shown is a maximum intensity projection, Hoechst (blue) indicates nuclei, TMRE (100 nm, yellow) indicates cells with ongoing respiration (mitochondrial transmembrane potential) and AnnV (Annexin V-FITC; 350ng / mL, green) indicates cells undergoing apoptosis. Prior to imaging the spheroids shown here were incubated in growth media for one-week postseeding. (B) Spheroids stained with TMRE (100 nM) to visualize viable cells in spheroids formed from 4000 MCF10A cells / well, in the indicated volume of media were imaged with a 5x air lens. Spheroids shown here were imaged after 3-hour incubation with the paramagnetic salt solution. Decreasing the volume of the cell suspension can make it possible to add sufficient gel (in liquid form) so that dilution of the hydrogel in the salt solution does not prevent gelation and therefore, encapsulation of the spheroid. Figure 6A shows the organoids produced after one week of growth that were stained with TMRE (100nM, yellow), Annexin V-FITC (350ng / ml_, green), and Hoechst 33342 (1 ug / ml, blue). Some cells that were not incorporated into, or exited from the structure, ended up on the bottom of the well where they died (green cells, apoptosis; blue nuclei without green or yellow cell bodies, died by an unknown mechanism). The main organoid is primarily TMRE positive (metabolically active) and not green (Annexin negative, alive). Spheroids were generated with the magnetic device from a 50 pL suspension of 4000 MCF10A cells containing 25pM paramagnetic salt, plated in wells of a 384 well imaging plate coated with polyHEMAto prevent adhesion during aggregation. The spheroids are from the same experiment shown in Figure 4A). Imaging used the PreciScan at 5x to locate the organoids because they moved around during handling and then the organoids were automatically reimaged at 20x air. Scale bar 100 .m.

[0092] Figure 6B shows images of spheroids of OVCAR8 cells (an ovarian cancer cell line). A 20 pL solution containing 4000 cells in paramagnetic salt was added to each well of a nonadherent imaging plate, and the cells in each well were formed into a spheroid with the magnetic device. Then the spheroids were washed and stained with TMRE (100nM, yellow) and MTC (1 .M, red). The indicated gel (PCT-Gel3 or Collagen) was added (Day 0) and the spheroids were imaged on day 7 with a 5X air lens. The process is benign as shown by the yellow staining indicative of metabolically active cells and the paucity of red staining indicative of stressed cells.

[0093] Figure 7 shows images of movement of MCF10A organoids by washing after they were generated on flat bottom non-adherent coated wells as also shown in Figure 4. Each panel is a composite of four micrographs of spheroids stained only with Hoescht 33342 (1 ug / ml, blue), that together provide an image of the entire well. When incubated in the salt solution on a non-adherent surface the cells aggregated in the magnetic field to generate a spheroid.

[0094] Figure 8A shows images of encapsulated spheroids derived from MCF10A. 4000 cells added as15 pL of cell suspension to each well were stained with TMRE (100 nM). PCT Gel 1 , a peptide modified PEG based hydrogel, was added to the wells at 35 pL per well (top 3 rows) or 30 pL per well (bottom 3 rows) (1) before media exchange at day 0. Each well is shown as a composite of four micrographs of spheroids stained only with TMRE. The stitching creates a faint black + that marks the center of the well. In the control wells without hydrogel (red squares), the spheroids moved towards the corner of the wells (lower righthand corner in the images) whereas the spheroids in the wells with hydrogel remained where they were initially formed, just off center (towards the upper left) in the wells; (2) after media exchange at day 0; close inspection reveals that the control spheroids (red squares) have changed position while the spheroids in wells containing the hydrogel have not moved. (3) when further handling was simulated by tilting the plate ~30 degrees and incubating overnight (After Tilt Day 1 , red squares) the spheroids moved from the right-hand to the lefthand side of the wells while the spheroids in the hydrogel containing wells (After Tilt Day 1 , no outlines) did not move.

[0095] Figure 8B shows images of encapsulated spheroids derived from OVCAR8 cells. 4000 cells were added in 20 pL of cell suspension to each well of a 384 well non-adherent imaging plate, formed into a spheroid with the magnetic device and washed and stained with TMRE (100nM, yellow) while the plate was affixed to the magnetic device. Then 20 pL of PCT-Gel1 was added (Day 0) to the indicated wells and after polymerization the media was exchanged on the indicated spheroids and then the plate was incubated on a tilt. The spheroids were imaged on Day 2, using a 5X air lens by confocal microscopy (Phenix Opera automated confocal microscope). Yellow staining indicates metabolic activity (alive cells).

[0096] Figure 9 shows images of encapsulated spheroids derived from MCF10A cells. 4000 cells as a 15 .L cell suspension, with or without dyes (Hoescht 33342 (1 ug / ml, blue), TMRE (100nM, yellow) and MTC (1 uM, red)) was added to the wells of a 384 well non-adherent imaging plate, formed into a spheroid with the magnetic device (3 hours), and then 35 pL of PCT-Gel1 was added and allowed to gel for 30 minutes. Then 20 pL of fresh media was added and washed away twice to remove salt. Finally fresh media with dyes was added. After 24 hours incubation the spheroids were located using Preciscan (5X air lens) and imaged at 20X with an air lens to visualize dye distribution. Even after 24 hours incubation, dye penetration was incomplete in the interior of the organoids for samples in which the dye was added after encapsulation (arrows).

[0097] Figure 10 shows progressive images of the proliferation of OVSRI-II patient derived organoids at day 7, day 22 and day 35. Micrographs are maximum intensity projections of organoids located using PreciScan at 5x followed by imaging at 20x with an air lens. Organoids were formed on Day 0 using the magnetic device by depositing 4000 cells in 15 pL of cell suspension to each well in polyHEMA coated (non-adherent) imaging plates with the dyes TMRE (100nM, yellow), Annexin V-FITC (350ng / ml_, green), MTC (1 uM, red). Once spheroids were formed, they were encapsulated with 35 pL of PCT-Gel1 . Yellow indicates metabolically active cells (growing), red indicates cell stress, white and green indicate dead and dying cells, respectively. Figure 11 A and B shows viability of encapsulated OVSRI-II patient derived organoids at day 41 from the same experiment as Figure 10, with the overnight addition of Hoescht 33342 (1 ug / ml, blue). (A) Micrographs are maximum intensity projections of organoids located using PreciScan at 5x followed by imaging at 20X with a water immersion lens. (B) Micrographs are maximum intensity projections of organoids located using PreciScan at 5x followed by imaging at 40X with a water immersion lens. Imaging at 40X enables visualization of individual energized mitochondria (TMRE, yellow) in the cytoplasm of the cells in the interior of the organoid. Unstained nuclei appear as dark areas in the yellow cytoplasm of cells in the interior of the organoid. Only portions of the organoids are shown as they are too big to fit in a single micrograph. Dyes added at Day 0 for images in A and B; TMRE (100nM, yellow), Annexin V-FITC (350ng / ml_, green), MTC (1 uM, red). Yellow indicates metabolically active cells (growing), red indicates cell stress, white and green indicate dead and dying cells, respectively.

[0098] Figure 12A-B show a maximum projection and cross-sectional view of an organoid grown in PCT-Gel4 (PEG based gel that has a crosslinking peptide that is degradable by matrix metalloproteases). The organoid was generated by depositing 4000 patient derived breast cancer cells (BB5 cells) in 15 .L of cell suspension to each well in polyHEMA coated (non-adherent) imaging plates, followed by spheroid formation using the magnetic device and encapsulating in 35 pL of PCT-Gel4 as above. The cells were grown in the plate at 37 °C in 5% CO2with regular media changes and imaged on day 27. (A) Micrographs are maximum intensity projections of organoids located using PreciScan at 5x followed by imaging at 20x with an air lens. (B) Micrographs are maximum intensity projections of organoids located using PreciScan at 5x followed by imaging at 40x with a water immersion lens. Dyes added at Day 0, for images in A and B; TMRE (100nM, yellow), Annexin V-FITC (350ng / ml_, green), MTC (1 uM, red). Nuclei were visualized by adding Hoescht 33342 (1 ug / ml, blue) overnight prior to imaging. Yellow indicates metabolically active cells (growing), red indicates cell stress, white and green indicate dead and dying cells, respectively.

[0099] Figure 13A-B shows a maximum projection and cross-sectional view of an organoid encapsulated in PCT-Gel4 that was imaged at incubation day 14. The organoid was generated from 4000 patient derived breast cancer cells (BB5 cells). The cells were formed into a spheroid by incubation in an agarose cup overnight followed by encapsulation in PCT- Gel4. (A) Micrographs are maximum intensity projections of organoids located using PreciScan at 5x followed by imaging with a 20x air lens.; (B) Micrographs are maximum intensity projections of organoids located using PreciScan at 5x followed by imaging with a 40x water immersion lens. Dyes were added at Day 0 for images in A and B; TMRE (100nM, yellow), Annexin V-FITC (350ng / ml_, green), MTC (1 uM, red). Nuclei were visualized by adding Hoescht 33342 (1 ug / ml, blue) overnight prior to imaging. Yellow indicates metabolically active cells (growing), red indicates cell stress, white and green indicate dead and dying cells, respectively.

[0100] With reference to the above Figures, unless specified otherwise, when the dyes were added, the concentrations and pseudo colors are as follows: TMRE (100nM, yellow), MTC (1 uM, red), Annexin V-FITC (350ng / ml_, green), Hoechst 33342 (1 ug / ml, blue).

[0101] Figure 14 shows representative images of Example 10; Maximum Projection of confocal Z-stack, 20x Water immersion lens, ChromaLive Yellow, ChromaLive Red, AnnexinV, Hoechst; Ovcar3-Day 13; Magnet plate.

[0102] Figure 15 shows representative images of Example 10; Maximum Projection of confocal Z-stack, 20x Water immersion lens, ChromaLive Yellow, ChromaLive Red, AnnexinV, Hoechst; Ovcar3-Day 13; Hydrogel cups.

[0103] Figure 16 shows representative images of Example 10; Maximum Projection of confocal Z-stack, 20x Water immersion lens, ChromaLive Yellow, ChromaLive Red, AnnexinV, Hoechst; Ovcar8-Day 13; Magnet plate.

[0104] Figure 17 shows representative images of Example 10; Maximum Projection of confocal Z-stack, 20x Water immersion lens, ChromaLive Yellow, ChromaLive Red, AnnexinV, Hoechst; Ovcar8-Day 13; Hydrogel cups.

[0105] Figure 18 shows representative images of Example 10; Maximum Projection of confocal Z-stack, 20x Water immersion lens, ChromaLive Yellow, ChromaLive Red, AnnexinV, Hoechst; LnCap-Day 13; Magnet plate.

[0106] Figure 19 shows representative images of Example 10; Maximum Projection of confocal Z-stack, 20x Water immersion lens, ChromaLive Yellow, ChromaLive Red, AnnexinV, Hoechst; LnCap-Day 13; Hydrogel cups.

[0107] Figure 20 shows representative images of Example 10; Maximum Projection of confocal Z-stack, 20x Water immersion lens, ChromaLive Yellow, ChromaLive Red, AnnexinV, Hoechst; PC3-Day 13; Magnet plate.

[0108] Figure 21 shows representative images of Example 10; Maximum Projection of confocal Z-stack, 20x Water immersion lens, ChromaLive Yellow, ChromaLive Red, AnnexinV, Hoechst; PC3-Day 13; Hydrogel cups.

[0109] Figure 22 shows an organoid grown from cells derived from a mesenteric metastasis of colon cancer: Z-stack of confocal micrographs of cells from Example 11 . Figure 23 shows an organoid grown from cells derived from a mesenteric metastasis of colon cancer Z-stack images reveal villus formation of cells from Example 11 .

[0110] Figure 24 shows an organoid grown from cells derived from a mesenteric metastasis of colon cancer Z-stack images reveal cell death and debris in villus formation of cells from Example 11.

[0111] Figure 25 shows an organoid grown from cells derived from a mesenteric metastasis of colon cancer Z-stack images of cells from Example 11- Chromalive provides information regarding death of cells, stress level of cells and debris formation.

[0112] Figure 26 shows an organoid grown from cells derived from a mesenteric metastasis of colon cancer Z-stack images reveal villus formation of cells from Example 11 .

[0113] Figure 27 shows organoid formation in hydrogel cups with different matrices. Figures 33-37 are higher magnification or confocal images of these organoids.

[0114] Figure 28 shows formation of primary human breast cancer organoids with hydrogel cups of the present invention.

[0115] Figure 29 shows confocal optical slices of primary human breast cancer organoids of Example 12 at Day 3; collagen I 100 pg / ml; 40x magnification.

[0116] Figure 30shows confocal optical slices of primary human breast cancer organoids of Example 12 at Day 3; Fibronectin 100 pg / ml; 40x magnification.

[0117] Figure 31 shows confocal optical slices of primary human breast cancer organoids of Example 12 at Day 3; iMatrix 50 pg / ml; 40x magnification.

[0118] Detailed Description

[0119] Definitions

[0120] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287- 9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8). Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the typical materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used.

[0121] As used herein, the term “compound” includes, without limitation, chemicals, pharmacological agents, small organic molecules, biomolecules, polypeptides, proteins, antibodies, sugars, polysaccharides, polynucleotides, cells, or combinations thereof. Such a compound may be a naturally-occurring product or a synthetic product.

[0122] As used herein, the term “cellular aggregate” refers to the 3D cell culture comprising spheroids or organoids. Unless it is explicitly stated to the contrary, the term “cellular aggregate” may comprise spheroids, organoids, or a combination of both (depending on their stage of development). Spheroids or organoids described herein can self-assemble or attach to one another to form a spheroid or an organoid, respectively. The cellular aggregate generally has a diameter in the range of about 100 to about 500 microns.

[0123] The term “organoid” is used herein to mean a 3-dimensional growth of mammalian cells in culture that retains characteristics of the tissue in vivo, e.g. prolonged tissue expansion with proliferation, multilineage differentiation, recapitulation of cellular and tissue ultrastructure, etc. Organoids are a sub-class of spheroids.

[0124] The term “spheroid” refers to a three-dimensional (3D) cluster of cells.

[0125] The term “hydrogel” is used in its conventional sense to refer to a material that absorbs a solvent (e.g. water), undergoes swelling without measurable dissolution, and maintains three-dimensional networks capable of reversible deformation. “Swelling” as referred to herein is meant the isotropic expansion of the hydrogel structure as water molecules diffuse throughout the internal volume of the hydrogel. The term hydrogel may include both desiccated and hydrated (e.g., solvent swollen) hydrogels.

[0126] The terms “peptide”, “polypeptide”, and “protein” as used herein have their plain and ordinary meaning as understood in light of the specification and refer to macromolecules comprised of amino acids linked by peptide bonds. The numerous functions of peptides, polypeptides, and proteins are known in the art, and include but are not limited to enzymes, structure, transport, defense, hormones, or signaling. Peptides, polypeptides, and proteins are often, but not always, produced biologically by a ribosomal complex using a nucleic acid template, although chemical syntheses are also available. By manipulating the nucleic acid template, peptide, polypeptide, and protein mutations such as substitutions, deletions, truncations, additions, duplications, or fusions of more than one peptide, polypeptide, or protein can be performed.

[0127] It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting. Many patent applications, patents, and publications are referred to herein to assist in understanding the aspects described. Each of these references are incorporated herein by reference in their entirety. When introducing elements disclosed herein, the articles “a”, “an”, “the”, and “said” are intended to mean that there may be one or more of the elements.

[0128] The term "comprising" and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and / or steps. The foregoing also applies to words having similar meanings such as the terms, "including", "having" and their derivatives. It will be understood that any embodiments described as “comprising” certain components may also “consist of’ or “consist essentially of,” these components, wherein “consisting of’ has a closed-ended or restrictive meaning and “consisting essentially of’ means including the components specified but excluding other components except for materials present as impurities, unavoidable materials present as a result of processes used to provide the components, and components added for a purpose other than achieving the technical effects described herein. For example, a composition defined using the phrase “consisting essentially of’ encompasses any known acceptable additive, excipient, diluent, carrier, and the like, suitable for the composition described herein. Typically, a composition consisting essentially of a set of components will comprise less than 5% by weight, typically less than 3% by weight, more typically less than 1% by weight of non-specified components.

[0129] It will be understood that any component defined herein as being included may be explicitly excluded from the claimed invention by way of proviso or negative limitation, such as any specific properties of the gellable material, certain components thereof, or method steps, whether implicitly or explicitly defined herein. For example, in aspects, the gellable material is not viscous.

[0130] In addition, all ranges given herein include the end of the ranges and also any intermediate range points, whether explicitly stated or not.

[0131] Finally, terms of degree such as "substantially", "about" and "approximately" as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least ±5% of the modified term if this deviation would not negate the meaning of the word it modifies.

[0132] The abbreviation, “e.g.” is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation “e.g.” is synonymous with the term “for example.” The word “or” is intended to include “and” unless the context clearly indicates otherwise.

[0133] The phrase “at least one of’ is understood to be one or more. The phrase “at least one of... and...” is understood to mean at least one of the elements listed or a combination thereof, if not explicitly listed. For example, “at least one of A, B, and C” is understood to mean A alone or B alone or C alone or a combination of A and B or a combination of A and C or a combination of B and C or a combination of A, B, and C.

[0134] Gellable Materials

[0135] Described herein is a gellable material for encapsulating a cellular aggregate. By “gellable material” it is meant that the material used for encapsulating the cellular aggregate is able to gel, or that gelation thereof can be triggered. In aspects, the encapsulating takes place in a well plate, such that the cellular aggregate can be subsequently imaged or used for screening purposes, for example. In addition, in aspects, by being encapsulated, the cellular aggregate has substantially no movement in the well plate during media exchange, handling of the well plate or tilting of the well plate. In other aspects, the cellular aggregate, once encapsulated, has no movement in the well plate during media exchange, handling of the well plate or tilting of the well plate. In other aspects, there is no movement in the well and in further aspects, there is limited movement in the well plate. In this way, by encapsulating the cellular aggregate described herein, a position of the encapsulated cellular aggregate in the well plate can be more readily determined. This can facilitate imaging or screening of the encapsulated cellular aggregate described herein.

[0136] As would be understood, the screening typically comprises adding a test compound to the cellular aggregate and determining the effect of the test compound on the cellular aggregate. Screening may comprise, for example, performing a drug screen, gene editing screen or RNA interference screen. To this end, the proliferation, growth, apoptosis or viability of the cellular aggregate, protein production, metabolic activity of key enzymes, expression of one or more genes (such as e.g. stress response genes), or the ability of the cellular aggregate to perform one or more functions may be determined in the presence relative to the absence of the test compound. For example, a decrease in proliferation, growth, viability or ability to perform one or more functions may be indicative that the compound has a toxic effect on the cellular aggregate, whereas an increase in the same may be indicative that the compound has a beneficial effect on the cellular aggregate. In aspects, the screening may be high throughput screening assays. The term “high throughput screening” as used herein refers to automated in vitro testing of the effect of compounds or conditions on cells and such screening is typically performed with the aid of computer or robot-controlled processes. Alternatively, and / or additionally, once the cellular aggregate is encapsulated, one would be able to determine, using an imaging microscope, for example, the position of the encapsulated cellular aggregate based on its relative positioning in x, y, and z axis of the imaging microscope. In this way, once the cellular aggregate is encapsulated, one would be able to locate the encapsulated cellular aggregate using the imaging microscope. Moreover, and when used in conjunction with a magnetic device (described in further detail below), the cellular aggregate, once encapsulated, is typically at the position predefined by the magnetic device, which can facilitate locating and imaging of the cellular aggregate. In aspects, and in respect of aiding the visualization of the cellular aggregate, the position is typically at a bottom of the well plate, which can allow for better imaging of the cellular aggregate with an inverted microscope, for example.

[0137] As further exemplified below, because the cells do not attach to the well plate, the cells do not remain in the center of the wells. This can be make locating the cellular aggregate and therefore imaging of the same difficult. In addition, during normal handling the cell aggregates roll around and often contact and stick to the walls of the wells. Again, this is undesirable because it adds significantly to the imaging time if the microscope must first ‘find’ the cellular aggregate. Moreover, if the cells adhere to the walls of the wells they will grow on the walls in a monolayer and may not produce the cellular aggregate (e.g. spheroid or organoid) desired for, for example, imaging. Surprisingly and advantageously, the gellable material described herein can eliminate these problems by holding the cellular aggregate in place. Additionally, the gellable material described herein can also provide an appropriate extracellular matrix to, for example, enable the spheroid to reorganize and form an organoid. The gellable material described herein can vary in respect to amount used, type of gellable material, etc, depending on, for example, the composition of the cells in the cellular aggregate (e.g. spheroids or organoids) described herein.

[0138] In aspects, the gellable material comprises a gellable substance, such as those selected from, but not limited to, collagen, fibronectin, Vitrogel™, iMatrix™, MD, or MC. In aspects, MD or MC comprise Collagen I, iMatrix, and Fibronectin. Collagen I, iMatrix, and Fibronectin can be at any suitable amount, such as for example, about 1 pg / ml to about 1000 pg / ml. Thus, Collagen I, iMatrix, and Fibronectin can be in an amount of about 1 pg / ml, about 10 pg / ml, about 20 pg / ml, about 30 pg / ml, about 40 pg / ml, about 50 pg / ml, about 60 pg / ml, about 70 pg / ml, about 80 pg / ml, about 90 pg / ml, about 100 pg / ml, about 150 pg / ml, about 200 pg / ml, about 250 pg / ml, about 300 pg / ml, about 350 pg / ml, about 400 pg / ml, about 450 pg / ml, about 500 pg / ml, about 550 pg / ml, about 600 pg / ml, about 650 pg / ml, about 700 pg / ml, about 750 pg / ml, about 800 pg / ml, about 850 pg / ml, about 900 pg / ml, about 950 pg / ml, or about 1000 pg / ml, or any number therebetween. In typical aspects, for MD, Collagen I is at an amount of about 500 pg / ml; iMatrix is at an amount of about 50 pg / ml; and is in an amount of about Fibronectin about 50 pg / ml. In typical aspects, for MC, Collagen I is in an amount of about 1000 pg / ml; iMatrix is in an amount of about 100 pg / ml; and Fibronectin in an amount of about 100 pg / ml.

[0139] In aspects, the gellable material described herein comprises a non-protein hydrogel. The non-protein hydrogel can be any suitable non-protein hydrogel capable of encapsulating the cellular aggregate as described herein. The non-protein hydrogel can be selected from, but is not limited to, polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), polyglycolic acid (PGA), polyvinyl alcohol (PVA), hyaluronan (HA) gels (e.g. semisynthetic TG cross-linked hyaluronan (HA) gel), alginate gels (e.g. natural calcium cross-linked alginate gel), and fibrin gels (e.g. human-derived thrombin cross-linked fibrin gel). In typical aspects, the non-protein hydrogel comprises PEG, and in further typical aspects, the PEG is a modified PEG. Modification of PEG is typically with a peptide, such as, for example, a dangling peptide or a crosslinking peptide. To this regard, and in aspects, the modified PEG comprises a PEG crosslinked by a peptide. For example, the modified PEG may be transglutaminase (TG) cross-linked PEG, a functionalised PEG (e.g. PEG modified with a fibronectin derived RGD peptide, laminin-derived peptides, fibronectin-derived peptides containing both the RGD motif and the PHSRN synergy site, or collagen l-derived peptide), PEG-alginate matrices, and the like.

[0140] In other aspects, the gellable material described herein comprises a protein hydrogel. The protein hydrogel can be any suitable protein hydrogel capable of encapsulating the cellular aggregate as described herein. The protein hydrogel can be selected from, but not limited to, collagen, elastin, gelatin, fibrin, fibronectin, or combination thereof. In typical aspects, the protein hydrogel comprises collagen. It would be understood that the properties of the hydrogels described herein may be modulated as desired, by varying the amounts of component(s), ratios of component(s), the density of component(s), etc., so long as the hydrogel is capable to encapsulating the cellular aggregate as described herein.

[0141] In aspects, the gellable material described herein is degradable. In this way, the gellable material described herein, as a hydrogel, for example, can be degraded by the cells so that the cellular aggregate (e.g. spheroid or organoid) can expand in size. Alternatively, the degradable gellable material may be removed from the encapsulated cellular aggregate such that the cellular aggregate may be used for further downstream activities (e.g. further biological testing). In aspects, the gellable material described herein has a low viscosity. In aspects, reference to the low viscosity is generally in relation to that of water. In addition, it is noted that if the gellable material is too viscous, the cellular aggregate may fall apart and / or the gellable material may not be able to encapsulate (e.g. hold) the cellular aggregate in place as described herein. Thus, in aspects, the gellable material described herein is not viscous. In typical aspects, the viscosity is similar to water prior to crosslinking. As such, in typical aspects, the viscosity is about 0.5 mPa.s to about 10 mPa.s, such about 0.5 mPa.s, about 0.6 mPa.s, about 0.7 mPa.s, about 0.8 mPa.s, about 0.9 mPa.s, about 1 mPa.s, about 1.5 mPa.s, about 2 mPa.s, about 2.5 mPa.s, about 3 mPa.s, about 3.5 mPa.s, about 4 mPa.s, about 4.5 mPa.s, about 5 mPa.s, about 5.5 mPa.s, about 6 mPa.s, about 6.5 mPa.s, about 7 mPa.s, about 7.5 mPa.s, about 8 mPa.s, about 8.5 mPa.s, or about 9 mPa.s, about 9.5 mPa.s to about 0.6 mPa.s, about 0.7 mPa.s, about 0.8 mPa.s, about 0.9 mPa.s, about 1 mPa.s, about 1.5 mPa.s, about 2 mPa.s, about 2.5 mPa.s, about 3 mPa.s, about 3.5 mPa.s, about 4 mPa.s, about 4.5 mPa.s, about 5 mPa.s, about 5.5 mPa.s, about 6 mPa.s, about 6.5 mPa.s, about 7 mPa.s, about 7.5 mPa.s, about 8 mPa.s, about 8.5 mPa.s, about 9 mPa.s, about 9.5 mPa.s, or about 10 mPa.s. In typical aspects, the viscosity is about 0.5 mPa.s to about 1.5 mPa.s, such as about 1 mPa.s.

[0142] In aspects, the gellable material described herein has a convenient gelling time to allow for the encapsulation of the cellular aggregate. Thus, the convenient gelling time can allow for reduction in the time required for the gellable material described herein to gel, and thus encapsulate the cellular aggregate. In this way, further downstream activities, such as imaging, can take place faster after the encapsulation process begins. In aspects, the gelling time is between about 1 minute to about 180 minutes, such as about 1 minute, about 3 minutes, about 5 minutes, about 7 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 70 minutes, about 75 minutes, about 80 minutes, about 85 minutes, about 90 minutes, about 95 minutes, about 100 minutes, about 105 minutes, about 110 minutes, about 115 minutes, about 120 minutes, about 125 minutes, about 130 minutes, about 135 minutes, about 140 minutes, about 145 minutes, about 150 minutes, about 155 minutes, about 160 minutes, about 165 minutes, about 170 minutes, or about 175 minutes, to about 3 minutes, about 5 minutes, about 7 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 70 minutes, about 75 minutes, about 80 minutes, about 85 minutes, about 90 minutes, about 95 minutes, about 100 minutes, about 105 minutes, about 110 minutes, about 115 minutes, about 120 minutes, about 125 minutes, about 130 minutes, about 135 minutes, about 140 minutes, about 145 minutes, about 150 minutes, about 155 minutes, about 160 minutes, about 165 minutes, about 170 minutes, about 175 minutes, or about 180 minutes. In typical aspects, the gelling time is about 10 minutes or about 30 minutes for the non-protein hydrogel described herein. In other typical aspects, the gelling time is about 60 minutes to about 180 minutes for the protein hydrogel described herein.

[0143] The well plate described herein can be any suitable well plate for imaging. In aspects, the well plate is a flat bottom well plate. The flat bottom well plate is typically preferred for screening. In other aspects, the well plate is a rounded bottom well plate. In other aspects, the well plate is made non-adherent with a non-adherent coating to assist with the prevention of cell adhesion to the well plate. The non-adherent coating can be a covalently bound hydrogel layer selected from Corning™ ultralow attachment range, Thermo Fisher™ Nunclon Sephera range, or polyHEMA (poly-2-hdroxylethyl methacrylate). In typical aspects, the non-adherent coating comprises polyHEMA.

[0144] The cellular aggregate can mature into an organoid or spheroid. In aspects, the cellular aggregate can mature into an organoid or spheroid prior to encapsulation described herein or after encapsulation described herein. In typical aspects, the cellular aggregate can mature into an organoid or spheroid after encapsulation described herein such that the subsequently matured organoid or spheroid can be held in place by the gellable material described herein and subsequently the position thereof can be determined.

[0145] In aspects, the cellular aggregate, before or after encapsulation, can mature into an organoid or spheroid for up to about 50 days, such as up to about 5 days, up to about 10 days, up to about 15 days, up to about 20 days, up to about 25 days, up to about 30 days, up to about 35 days, up to about 40 days, up to about 45 days, or up to about 50 days. In aspects, the cellular aggregate, before or after encapsulation, can mature into an organoid or spheroid for about 42 days. Methods of maturing and / or growing organoids and spheroids would be understood by the skilled person and are further described in the method section below.

[0146] The cells of the cellular aggregate described herein can be derived from any suitable cellular source, including but not limited to, cells from primary tissue, including cancerous and non-cancerous tissue, stem cells, pluripotent stem cells or tumour cells such as a primary tumour cell or a tumor cell line. For example, primary tissue may be derived from the colon (including healthy and colorectal carcinoma), pancreas (including healthy and pancreatic cancer), oesophagus (including healthy and oesophageal cancer), stomach (including healthy and stomach cancer), intestine (including healthy and bowel cancer), liver (including healthy and liver cancer), prostate (including healthy and prostate cancer), mammary gland (including healthy and breast cancer), biliary tree (also known as biliary tract, and including healthy and cancer biliary epithelium). Thus, in aspects, the organoid or spheroid is produced from cells derived from various tissues or organs, such as skin, breast, lung, liver, pancreas, kidney, blood, stomach, intestine, spleen, ovary, heart, cervix, bladder, oral cavity, esophagus, prostate, or mesentery. In typical aspects, the cellular aggregate or the encapsulated cellular aggregate described herein is an organoid or a spheroid, which has been derived from the aforementioned sources.

[0147] Methods of Use of the Gellable Materials

[0148] Described herein, in aspects, is a method for producing an encapsulated cellular aggregate in a well plate. In aspects, the method comprises adding the gellable material described herein to the cellular aggregate described herein, and forming the encapsulated cellular aggregate by gelling the gellable material described herein. Similar to the product (gellable material) described above, the method described herein is suitable for producing the encapsulated cellular aggregate that can be used for imaging and / or screening since the encapsulated cellular aggregate is held in place (e.g. substantially no movement thereof) as described above. For the sake of brevity and to avoid repetition, similar elements between the product (gellable material) and method sections of the application, such as, the cellular aggregate and the gellable material will not be described since the same description is applicable to both sections.

[0149] In aspects, the adding comprises diffusing the gellable material over a period of time. The diffusing typically comprises adding the gellable material to the well plate gently so as to not damage the cellular aggregate. This can avoid not only disruption of the cellular aggregate, but also prevent the cellular aggregate from forming a monolayer on the bottom of the well plate, either of which would hinder the ability to locate or image the cellular aggregate. In aspects, the diffusing comprises layering the gellable material. In this way, the gellable material described herein can mix with the culture media around the cellular aggregate in the well plate. Thus, the diffusing can allow for suitable mixing of the gellable material and the culture media without the need for mechanical mixing which could, for example, damage the cellular aggregate. In aspects, the gellable material is added at concentrations higher than those required for gelation such that when the gellable material mixes with the culture media surrounding the cellular aggregate, the final concentration is sufficient for gelation of the gellable material to occur. In aspects, the period of time is about 1 minute to about 180 minutes, such as about 5 minutes, about 15 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes, about 75 minutes, about 85 minutes, about 90 minutes, about 100 minutes, about 110 minutes, about 120 minutes, about 130 minutes, about 140 minutes, about 150 minutes, about 160 minutes, about 170 minutes, or about 180 minutes. For example, the period of time for diffusion of the nonprotein hydrogel described herein can be about 10 minutes or 30 minutes, and the period of time for the diffusion of the protein hydrogel described herein can be about 60 minutes to about 180 minutes. The period of time is typically sufficient to allow the gellable material to solidify such that the encapsulated cellular aggregate with minimal movement (e.g. with the plate titling / handling as described herein), is thus formed. With the reduced amount of time of diffusion, the time to encapsulate the cellular aggregate described herein and subsequently locate and image the same, is typically reduced by the method described herein.

[0150] Methods of forming the cellular aggregate described herein are further described in the examples provided below. These methods can be carried out magnetically on flat nonadherent surfaces, where a salt solution (described below) is added to the well plate and the well plate is placed on the magnetic device in an incubator to allow for aggregation. Alternatively, cellular aggregation can be carried out using a non-adherent surface that has a refractive index close to that of the cells. In the latter method, a non-adherent hydrogel, such as agarose, which is sufficiently viscous to form a curved surface (described in further detail below) may be used. These methods are described in greater detail below. Following formation of the cellular aggregate by either of the above-noted methods, the gellable material described herein can be added thereto to allow for encapsulation thereof.

[0151] Thus, in aspects, the method described herein further comprises growing cells into the cellular aggregate prior to the adding the gellable material. For example, the cells may be grown in a monolayer culture in culture media. In aspects, the cells are obtained from tissue that has been disaggregated into a cell suspension which can then be cultured by methods understood by the skilled person. To this regard, and as would be understood, the growing, proliferation and / or maturation of cells and / or the cellular aggregates described herein can be obtained by using culture medium, such as a composition that comprises at least nutrients and additional factors that support said growing, proliferation and / or maturation (such as e.g. growth factors, mitogens and pathway modulators). In typical aspects, the culture medium is a liquid composition comprising at least one of the aforementioned factors. In aspects, the culture medium may be a chemically defined medium, such as a nutritive solution for culturing cells which contains only specified components, preferably components of known chemical structure. The chemically defined medium is typically devoid of undefined components or constituents which include undefined components, such as feeder cells, stromal cells, serum, serum albumin and complex extracellular matrices, such as Matrigel™. In aspects, the chemically defined medium may be humanised. A humanised chemically defined medium is typically devoid of components or supplements derived or isolated from non-human animals, such as Foetal Bovine Serum (FBS) and Bovine Serum Albumin (BSA), and mouse feeder cells. In aspects, the culture medium may be conditioned medium which includes undefined components from cultured cells and is not chemically defined. In other aspects, the culture medium may be a concentrated culture medium in which a composition is designed to be diluted prior to being used as a culture medium, for example using water (distilled or sterilised, as appropriate) or a buffer. In other words, a concentrated version of a culture medium is a composition that contains the ingredients of a culture medium in concentrations higher than those intended for use as a culture medium.

[0152] The culture medium typically comprises a basal medium, including, but not limited to, Iscove's Modified Dulbecco's Medium (IMDM), Ham's F12, Advanced Dulbecco's modified eagle medium (DMEM) or DMEM / F12. In typical aspects, the basal medium is advanced DMEM. In addition, the basal medium may be supplemented with a media supplement, such as N2(Gibco), B-27™ (ThermoFisher) and / or one or more additional supplements which may include L-glutamine or substitutes, such as L-alanyl-L-20 glutamine (e.g. Glutamax™), nicotinamide, N-acetylcysteine, buffers, such as HEPES, and antibiotics such as blasticidin or puromycin. For example, the basal medium (e.g. advanced DMEM) may be supplemented with HEPES, Glutamax, N2 and optionally nicotinamide and / or N-acetylcysteine. Such a medium may be a chemically defined medium.

[0153] Additional factors that support proliferation and / or differentiation of the cellular aggregate described herein may also be included in the culture medium described herein. These may include pathway modulators, vitamins and tropic mitogens, such as, for example, growth factors (such as epidermal growth factor (EGF), fibroblast growth factor 10 (FGF10)), a TGFp inhibitor, a non-canonical Wnt signalling potentiator, a BMP inhibitor, hormones (such as e.g. gastrin and / or prostaglandin E2), a canonical Wnt ligand, and a p38 MAPK signalling inhibitor. The addition of said mediators, including which mediators and the appropriate amounts thereof, would be understood by the skilled person.

[0154] In other aspects, the gelling of the gellable material allows for the encapsulating of the cellular aggregate described herein. In this way, the gellable material described herein can gel to surround the cellular aggregate described herein so that the cellular aggregate can be encapsulated by the gellable material described herein when the gellable material described herein solidifies. In aspects, the gelling of the gellable material occurs for a period of time, such as, for example, about 1 minute to about 180 minutes as described above. As also described above, the non-protein hydrogel typically gels for about 10 minutes or 30 minutes, and the protein hydrogel typically gels for about 60 minutes to about 180 minutes.

[0155] In aspects, the method further comprises adding a dye compound to the well plate prior to the adding the gellable material. In other aspects, the method further comprises adding a dye compound to the well plate after the forming the encapsulated cellular aggregate. In aspects, the dye may be mixed with the gellable material and then added to the growing cells or to the encapsulated cellular aggregate. The dye compound can allow for detecting or monitoring changes in cell state (e.g., cell physiology, behavior) in response to an agent or a stimuli such as changes in pH, temperature, salt concentration, contact with other cells, or treatment with a drug or drug candidate.

[0156] In aspects, the dye compound is selected from, but not limited to:

[0157] TMRM (Tetramethylrhodamine methyl ester),

[0158] TMRE (Tetramethylrhodamine ethyl ester),

[0159]

[0160] 5

[0161] Hoescht 33342, or a salt thereof.

[0162] In other aspects, the method further comprises adding a drug to the encapsulated cellular aggregate. The drug may be any compound or composition thereof having a preventative or therapeutic effect, including and without limitation, antibiotics, peptides, hormones, organic molecules, vitamins, supplements, factors, proteins and chemoattractants. In typical aspects, the drug is an anti-cancer drug.

[0163] In other aspects, the method further comprises growing cells, such as fibroblasts, on the encapsulated cellular aggregate. In other aspects, the method further comprises maturing the cellular aggregate, before or after encapsulation, into spheroids over a period of time. In other aspects, the method further comprises maturing the cellular aggregate, before or after encapsulation, into organoids over a period of time. As described above, the typical period of time for said maturation is up to about 50 days, such as up to about 35 days, up to about 40 days, up to about 45 days, or up to about 50 days. In further typical aspects, the cellular aggregate, before or after encapsulation, is the spheroid or the organoid described herein. The spheroid or the organoid may be derived from any of the sources as described herein. In other aspects, the method further comprises adding a paramagnetic agent to the well plate prior to the adding the gellable material. To this regard, the preparation of the cellular aggregates involves the use of a magnetic device, such as that which is described in US Patent No. 11788057, hereby incorporated by reference in its entirety. The paramagnetic agent may be any suitable paramagnetic agent, but in typical aspects, the paramagnetic agent comprises a Gadolinium based salt or contrasting agent, optionally Gadoliniumdiethylenetriamine penta-acetic acid (Gd-DTPA). The paramagnetic agent may be prepared in a solution that is suitable for the cellular aggregate to form in the well plate. For example, in aspects, the solution may comprise Gd-DTPA mixed with phosphate-buffered saline or culture medium. A skilled person would readily be able to identify other buffers or culture media suitable for use depending on the cellular aggregate to be formed.

[0164] In aspects, the paramagnetic solution is added at a concentration of about 25 pM. The concentration of paramagnetic agent used in the methods described herein may be higher or lower depending on the nature of the conditions and the nature of the cells / cellular aggregate. For example, the concentration of the paramagnetic agent may depend on factors such time, the size and type of cellular aggregate, and magnetic factor considerations between the device and the paramagnetic solution. In typical aspects, after the paramagnetic agent is added to the well plate, a further media exchange is conducted to remove the salt solution from the gellable material. This is again, typically by diffusion, as the gellable material by this time has now solidified and there is therefore no mixing of the medium and the gellable material. The magnetic device can be placed in an incubator and after a period of time, such as about 3 hours incubation, the gellable material described herein can be added to the well plate, to which point the well plate can be removed from the magnetic device and the gellable material is allowed to gel as described herein. This can prevent the cellular aggregate (e.g. spheroid) from smearing into the solidified gellable material. In other aspects, the well plate can remain in the magnetic device to allow the gellable material to gel, after which the well plate can be removed therefrom.

[0165] Similar to the gellable material description above, the well plate can be a flat bottom well plate or a round bottom well plate. In aspects, as was described for the gellable material above, the flat bottom well plate may comprise a non-adherent coating selected from Corning™ ultralow attachment range, Thermo Fisher™ Nunclon Sephera range, or polyHEMA (poly-2-hdroxylethyl methacrylate).

[0166] In aspects, as has been described herein, the gellable material can be any gellable substance, such as, but not limited to, collagen, fibronectin, Vitrogel™, iMatrix™, MD, or MC. In addition and as described above, the gellable material may comprise the protein or nonprotein hydrogels as described herein.

[0167] In other aspects, it is possible to use round bottomed non-adherent well plates, harvest the cellular aggregate (e.g. spheroids) with a pipettor, then transfer them to a new multi-well imaging plate that has liquid extracellular matrix (ECM) (e.g. Matrigel™) in the wells and then trigger gelation. The potential difficulty with this method surrounds triggering gelation because it has to be at a time when the cellular aggregate (e.g. spheroid) has entered into the gellable material but before it sinks to the bottom of the well. This could make it very difficult to know where the cellular aggregate (e.g. spheroids / organoids) are in the well plate; particularly in Z. In addition, this can be made even more complicated by putting a thin layer of hydrogel in the bottom of the wells transferring in a spheroid and then adding liquid hydrogel and triggering gelation.

[0168] In order to overcome some of these problems, in aspects, a method of forming a non-adherent hydrogel cup for cellular aggregate formation is provided. The method comprises dispensing a non-adherent hydrogel to a well plate at a temperature. The non- adherent hydrogel is sufficiently viscous to form the non-adherent hydrogel cup having a curved surface. Thus, in aspects, a shape of the non-adherent hydrogel cup can be determined by the viscosity of the non-adherent hydrogel while in liquid form. In other words, the method, in aspects, comprises using the viscosity of the non-adherent hydrogel to form a cup that can be used to form and then support growth / differentiation of the cellular aggregate without having the transfer step described above. In this method, the non-adherent hydrogel cup can be formed by the temperature dependency of the viscosity of the liquid form of the non-adherent hydrogel. In aspects, the higher the viscosity of the non-adherent hydrogel, the steeper the sides of the cup that is formed. In aspects, the temperature used to form the cup is about 45°C to about 70°C, such as about 45°C, about 50°C, about 55°C, about 56°C, about 57°C, about 58°C, about 59°C, about 60°C, about 61 °C, about 62°C, about 63°C, about 64°C, about 65°C, about 66°C, about 67°C, about 68°C, about 69°C or about 70°C. In typical aspects, the temperature is about 60°C. In aspects, once the non-adherent hydrogel is dispensed into the well plate, the method further comprises allowing the non-adherent hydrogel to cool. This can facilitate the formation of the shape of the non-adherent hydrogel cup. In addition, these non-adherent cups are typically formed in well plates that have a flat imaging bottom and rounded wells. In typical aspects, the well plate does not have square wells. While the non-adherent hydrogel may be any suitable non-adherent hydrogel that typically has a refractive index close to that of the cells, in typical aspects, the non-adherent hydrogel is agarose. Once cellular aggregates are formed in the non-adherent hydrogel cup, the cellular aggregate maybe encapsulated by the gellable material and / or methods described herein. This hydrogel cup may be better for imaging than the round bottom low adhesion plastic because there is less distortion of the image. As provided by the examples below, however, agarose wells may require more calculation in the PreciScan sequence since a slight change in agarose deposition or organoid position can result in a relatively large difference in z position. As such, the flat bottom well plates (with gellable material encapsulation as described herein) can allow for a faster collection of data.

[0169] In specific aspects, involving the non-adherent hydrogel cup described above and exemplified below, the preparation of the cellular aggregates (e.g. spheroids) typically comprises adding the about 500 to about 5000 cells the cups and then centrifuging the plate for about 30 seconds at 300rpm. The well plate may be then rotated about 180 degrees and subjected to a second spin for about 30 seconds at 300 rpm. Thus, the cells are typically centrifuged into the bottom of the non-adherent hydrogel cup to which point the cells are incubated to allow the cells to aggregate for about 12 hours to about 24 hours. In typical aspects, after the incubation of the cells, the gellable material described herein, can be added to the non-adherent hydrogel cups to encapsulate the formed cellular aggregates, after gelation of the gellable material takes place.

[0170] Also described herein is a kit for encapsulating cellular aggregates as described herein. In typical aspects, the kit comprises the gellable material described herein. The kit may further comprise any one or all of the following: the culture medium, water, distilled water, sterilised water, buffer or instructions for use. The kit may further comprise one or more fluorescent dyes or cell culture containers, such as well plates or flasks, and in typical aspects, the well plates comprise non-adherent coating, such as polyHEMA.

[0171] The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific Examples. These Examples are described solely for purposes of illustration and are not intended to limit the scope of the invention. Changes in form and substitution of equivalents are contemplated as circumstances may suggest or render expedient. Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the constructs of the present invention and practice the claimed methods. The following working examples therefore, specifically point out the typical aspects of the present invention and are not to be construed as limiting in any way in the remainder of the disclosure. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation. EXAMPLES

[0172] Example 1 Generation of organoids using hydrogel cup or flat bottom well plates

[0173] Organoids were generated from 4000 MCF10A cells per well using hydrogel (low melting agarose) cups (rounded bottoms) formed on flat bottom wells or flat bottom wells with a nonadherent polyHEMA coating. Both hydrogel cups and flat bottom wells can be useful for imaging or screening.

[0174] Methods: Organoid production - applies to all examples:

[0175] 1 . Preparation of plates. For spheroid generation using the magnetic device, about 20 .L / well of about 20 mg / mL polyHEMA in about 95% ethanol was added to 384 well-plates and allowed to air dry overnight. Then the culture plate was positioned on the magnet device in a biosafety hood.

[0176] To form non-adherent hydrogel cups, low melting temperature agarose was added at about 7 to about 10 .L / well at about 45°C to about 60°C, depending on the desired curvature, and allowed to cool to room temperature. The lower the temperature at the time of dispensing into the wells, the higher the viscosity, and therefore the steeper the sides of the cup. In Figure 1 , the temperature used was about 60°C.

[0177] 2. Preparation of spheroids: about 4000 cells / well were added to the wells with or without dyes as specified in the figure captions. For preparation of spheroids in agarose cups, the cells were added to the cups and the plate was centrifuged briefly (about 300rpm for about 30 seconds, then the plate was rotated about 180 degrees and subjected to a second spin of about 300 rpm for about 30 seconds) and then placed in a 37°C incubator with 5% CO2and spheroids formed during an overnight incubation. Where specified, after overnight incubation, hydrogel (PCT-Gel 4) was added to the agarose cups to encapsulate the spheroids. For preparation of spheroids with the magnetic device, the salt solution was added to a concentration of about 25 pM, and the plate placed on the magnetic device in an incubator. After about 3 hours incubation, the PCT-Gel or collagen solution was added to the plate and it was removed from the magnetic device and allowed to gel. This can prevent smearing of spheroids into the solidified gel. PCT-Gels gel in approximately 10 minutes. In other experiments the plate remained on the magnetic device to allow the solution to gel and then the plate was removed. PCT-Gels were allowed to solidify for about 30 minutes. Collagen gels solidify in about 1 to about 3 hours depending on the concentration. Fresh media was added for about 10 to about 30 minutes, then replaced twice to remove excess salt, and then fresh media with or without dyes, as specified, was added and the plate returned to the incubator.

[0178] 3. Addition of dyes. The dyes specified in the figure captions were added as follows and are presented in the images with the indicated pseudo color: TMRE (about 100nM, yellow), MTC (about 1 uM, red), Annexin V-FITC (about 350ng / ml_, green), Hoechst 33342 (about 1 ug / ml, blue). In Figure 1 only TMRE was added.

[0179] Results: As shown in Figure 1 , forming spheroids in the flat bottom wells with the magnetic device tended to pack the cells together more tightly than incubation in the hydrogel cup. The medium yellow color of the TMRE indicates normal metabolism (mitochondrial transmembrane potential) in the cells. Both the magnetic method and the hydrogel cups led to spheroid formation at a relatively well-defined location in X, Y and Z. As a result, the search area was small which can enable rapid localization using the Preciscan function of the Phenix Opera.

[0180] Example 2 Microscopic image of hydrogel cup

[0181] Methods: Agarose cups were formed as described above in Example 1 . After cup formation, about 20,000 fluorescent beads were added to the well and allowed to settle for about 1 hour. A series of confocal micrographs were recorded at different Z-heights using the Phenix Opera automated confocal using a 5x air lens to enable visualization of the entire meniscus.

[0182] Results: As shown in Figure 2, using the meniscus formed from the liquid agarose enables creating a symmetrical well with the bottom less than about 300 pm from the ultrathin window of the wells of the imaging plate. In this example, it is noted that by making use of the viscosity of the hydrogel prior to gelation it was possible to pre-determine the geometry. After adding cells and letting them clump / form a spheroid, a second hydrogel can be added (as described in further examples) to both support growth and differentiation into an organoid and as well as to hold the spheroid / organoid in place. Reprogramming medium can encourage the cells to reorganize the initial spheroid into an organoid. Thus, the shape of the agarose cup can enable aggregation of the cells at a predefined location as a spheroid that will reorganize over time into an organoid. Example 3 Assessment of the addition of further agents to well plates and cell viability

[0183] In order to determine if the paramagnetic agent solution that the cells are incubated in to enable magnetic aggregation affected the viability of the cells in the wells, organoids were developed as described in Example 1 and the viability of the cells was assessed in the presence of the paramagnetic agent.

[0184] Methods: Organoids were produced from three different patient derived primary breast cancer cells (BBSRI-5, BBSRI-7, BBSRI-8) in the flat bottom well imaging plate using the magnetic device as described in Example 1. The paramagnetic agent used was gadoliniumdiethylenetriamine penta-acetic acid (Gd-DTPA).

[0185] Results: As shown in Figure 3, the paramagnetic agent (e.g. MRI contrast agent) did not cause an increase in TMRE staining indicative of ER stress to the cells. In general, addition of the paramagnetic agent also did not decrease staining by TMRE suggesting no loss in metabolic activity (mitochondrial transmembrane potential). However, it was noted that a small number of cells in BBSRI-8 had lower TMRE intensity but it is a very small fraction of the total cells. This was likely due to experimental variation (see, the white spot below the brown spot in the bottom right image of Figure 3). None of the data moves to the right indicating that the salt solution did not kill any cells (Annexin (AnnV) reports on cell death). Thus, it was found that the paramagnetic agent treatment does not significantly affect viability even when included in the media for about 1 week post treatment (see, also Figures 5A and 6).

[0186] Example 4 Non-adherent coating of well plates

[0187] Having shown that the viability of the cells was not impacted by the addition of a paramagnetic agent of Example 3, organoid development was further assessed using nonadherent coated well plates and non-coated well plates in the presence of the same paramagnetic agent.

[0188] Methods: Organoids were produced as described in Example 1 . Wells were either coated with a non-adherent coating (e.g. polyHEMA) or were uncoated as described in Example 1. The cells were stained with Hoescht 33342 to visualize cell nuclei and imaged by confocal microscopy. Nuclei appear as light blue to white depending on the amount of chromatin (~number of nuclei / image pixel). The time required to generate the organoid in the presence of the paramagnetic agent was also assessed for cells stained with TMRE to verify that the mitochondrial transmembrane potential remained intact.

[0189] Results: As shown in Figure 4A, in the control wells (no SALT, no coating) the cells grew as a monolayer on the bottom of the plate, as expected. When the paramagnetic agent was added (+SALT, no coating) the cells were moved towards the center of the well, as expected, but grew on the bottom of the well and began to spread out into a monolayer. When the cells were plated on a non-adherent surface with the paramagnetic agent (+SALT, +coating) the cells aggregated into spheroids (appear white on the dark blue background). As shown in the bottom row of Figure 4A, the spheroids that were produced did not adhere to the plate and therefore during normal handling moved to various positions in the well including adhering to the walls of the wells. Spheroids that contacted the walls of the wells can adhere because the walls of the wells do not have a non-adherent coating. Once attached to the wall the cells begin to form monolayers on the walls of the wells. This is undesirable as the end result will be a monolayer that cannot be imaged, instead of a 3D organoid.

[0190] Figure 4B shows that when the exposure time to the paramagnetic agent was assessed, only about 3 hours exposure to the paramagnetic agent on the magnetic device was needed to generate spheroids on non-adherent plates. The spheroids that formed in the paramagnetic agent on the non-adherent surface retained mitochondrial transmembrane potential characteristic of normal metabolic activity (normal TMRE intensity). Both spheroids (example wells labeled 3-4 hours time in the magnetic fields) and partially aggregated cells (example wells labeled 1-2 hours time in the magnetic fields) are not near the center of the wells. This indicates that in a plate with a non-adherent coating, the cells shifted position during normal handling.

[0191] Example 5 Different seeding volumes for organoid development in non-adherent coated wells

[0192] In order to determine if it was possible to reduce the liquid used to generate the spheroid / organoid such that the gellable material (e.g. hydrogel) described herein could be added to the well without it being diluted too much to gel, different volumes of cell suspensions in paramagnetic agent were tested to generate the spheroids using the magnetic device and viability was assessed.

[0193] Methods: Spheroids were generated as described in Example 1 . Different volumes of cell suspensions each containing about 4000 MCF10A cells in paramagnetic agent solution (about 15pL, about 20pL, about 30 pL, about 40 pL and about 50 pL) were used to add the cells to the wells in the imaging plates coated with a non-adherent coating (polyHEMA). After spheroids were formed using the magnetic fields, the plates were carefully removed from the magnetic device and the paramagnetic agent solution was replaced by carefully adding and then removing about 40 p,L of media twice using a Multiflo plate washer. The plate was then incubated at 37°C and 5% CO2 for about 1 week and imaged by spinning disc confocal microscopy on the Phenix automated microscope. Individual wells were manually selected for presentation in which the spheroids did not contact the walls of the wells.

[0194] Results: As shown in Figure 5A, changes in seeding volume (i.e. about 15pL to about 50 pL) resulted in no difference in organoids generated, as observed about 1-week postseeding. Organoids were stained with TMRE (about 100nM, yellow), Annexin V-FITC (about 350ng / ml_, green), Hoechst 33342 (about 1 ug / ml, blue). The blue stain identifies nuclei, the yellow indicates the mitochondrial transmembrane potential (metabolic activity) and green indicates dead or dying cells. Another image of the affect of decreasing the seeding volume is shown in Figure 5B for a larger number of spheroids stained only with TMRE (about 100nM, yellow) and imaged immediately after the about 3-hour incubation period. In the no salt control (about 50 p.L, -salt) spheroids were not formed, as expected.

[0195] Example 6 Organoid encapsulation

[0196] While spheroids / organoids could be generated using the flat bottom well plates with a nonadherent coating as described in the above Examples, the organoids had a tendency to roll around during handling which presented problems, for example, if the spheroid contacted the wall of the well and it adhered and generated a monolayer. In addition, movement of the spheroids / organoids can create problems for imaging due to the time required to locate each one. Therefore, it was assessed whether or not the spheroids / organoids could be held in place by using an encapsulation method. The exemplary cell used to generate the spheroids were MCF10A cells, a premalignant breast epithelial cell line.

[0197] Methods: Spheroids were developed from MCF10A cells on non-adherent coated plates of Example 4. Organoids were encapsulated as described in the figure captions. The gellable material (e.g. hydrogel) described herein was tested for its ability to hold the organoid in place such that it would not roll over to the left / right of the plate when the plate is tilted in the incubator at between about 30 to about 45 degrees overnight. As an additional advantage, the hydrogels used in the different figures (PCT-Gel1 , PCT-Gel4, Collagen) can all support, albeit to varying degrees, the differentiation of spheroids into organoids.

[0198] Results: The organoids generated as per Example 5 required the use special software (Preciscan) to control the microscope to find the organoids. This is related to the fact that, as shown in Figure 7, the organoids had a tendency to roll around (not stay in place). It was of interest to encapsulate the generated organoids so that they could remain in place during media exchange or after a tilt assay, for example. As shown in Figure 8A, when about 4000 MCF10A cells in about15 pL suspension were added to each well, in media containing TMRE (about 100 nM), they were encapsulated by adding about 35 pL of a peptide modified PEG based hydrogel (PCT-Gel1) before media exchange at day 0. PCT-Gel1 was added at about 3 hours and allowed to gel for about 30 minutes at about 37 °C and spheroids were imaged by confocal microscopy with a 5X air objective using the Phenix Opera automated microscope. Each panel is a composite of four micrographs of spheroids stained only with TMRE. The image stitching creates a faint black + that marks the center of the well. Panel 1) In the control wells without hydrogel (wells outlined with red squares), the spheroids moved towards the corner of the wells (lower right-hand corner in the images) whereas the spheroids in the wells with hydrogel remained where they were initially formed, just off center (towards the upper left) in the wells; Panel 2) after media exchange at day 0; close inspection reveals that the control spheroids without encapsulating hydrogel (red squares) have changed position while the spheroids in wells containing the hydrogel have not moved. Panel 3) when further handling was simulated by tilting the plate approximately 30 degrees in the incubator overnight (After Tilt Day 1 , red squares) the spheroids moved from the righthand side to the left-hand side of the wells while the spheroids in the hydrogel containing wells (After Tilt Day 1 , no outlines) did not move.

[0199] Figure 8B shows images of OVCAR8 cells, about 4000 cells in about 15 .L of suspension added to each well of a non-adherent imaging plate, formed into a spheroid with the magnetic device and stained with TMRE (about 100nM, yellow). The indicated gel (PCT- Gel 1 ) was added on Day 0 to the indicated wells and after polymerization (about 30 min) the media was exchanged for media alone or media containing PCT-Gel1 as indicated and then the plate was incubated on a tilt. The spheroids were imaged on Day 2, using a 5X air lens. In the absence of the gel, the spheroids were pushed to the sides of the wells by the media change and / or moved when incubated on a tilt. In contrast, the spheroids encapsulated in PCT-Gel1 remained near the center of the wells. Yellow staining indicates metabolic activity (alive cells). The results suggest that use of the magnetic device or agarose cup can be superior to alternative approaches in which round bottomed non-adherent plates are used to generate spheroids overnight or rapidly with the magnetic device and then the spheroids are harvested with a pipettor and transferred to a new multi-well imaging plate that has liquid ECM (matrigel or hydrogel) in the wells and gelation is triggered (just before, together with, or just after adding the spheroid). In this method timing is difficult to control because gelation must occur at a time when the spheroid has entered into the gellable material but before it sinks to the bottom of the well. In practice it is very difficult to control where the spheroids / organoids are located - particularly in Z. As a result, the time required for imaging is extended substantially. It is also much more expensive and time consuming to transfer spheroids between plates. Another complicated approach involves putting a thin layer of ECM hydrogel in the bottom of the wells, transferring in a spheroid made with the magnetic device and then adding liquid ECM or hydrogel and triggering gelation. In respect of this latter approach, it may be improved by using the viscosity of the liquid hydrogel to form a cup that can be used both to form and then to support growth / differentiation of the organoid thereby obviating the transfer step. The hydrogel cup is typically better for imaging than the round bottom low adhesion plastic because there is less distortion of the image due to the change in refractive index and the curved shape of the plastic.

[0200] Example 7 Staining encapsulated organoids with fluorescent dyes

[0201] The effect of encapsulation on staining organoids with fluorescent dyes was tested. The exemplary cells used were MCF10A cells, described in Example 6 and Figure 8A. Organoids were stained with non-toxic dyes before and after encapsulation in PCT-Gel1 to test dye penetration.

[0202] Methods / Results: About 15 pL cell suspension containing about 4000 MCF10A cells, with or without dyes (Hoescht 33342 (about 1 ug / ml, blue), TMRE (about 100nM, yellow) and MTC (about 1 uM, red)) was added to each well of a non-adherent imaging plate, formed into a spheroid with the magnetic device (about 3 hours), and then about 35 pL of PCT-Gel1 was added and allowed to gel for about 30 minutes. Then about 20 pL of fresh media was added and washed away twice to remove the paramagnetic salt. Finally, fresh media with dyes was added. After about 24 hours incubation, the spheroids were located using Preciscan (5X air lens) and confocal micrographs were recorded using a 20X air lens to visualize dye distribution. Even after about 24 hours incubation, dye penetration was incomplete in the interior of the organoids for samples in which the dye was added after encapsulation (Figure 9, arrows). Staining the organoids at the time of their generation, prior to encapsulation, can result in better stain penetration and therefore more even staining throughout the organoids with all dyes tested (Figure 9). The results shown suggest that non-toxic dyes can be added to the cells before encapsulation.

[0203] Example 8 Assessment of viability of encapsulated organoids

[0204] The viability of the encapsulated organoids, produced from different cell types, was tested. The exemplary cells used were MCF10A cells, described in Example 6, OVCAR8 (Figure 6B), OVSRI-II, primary human ovarian cancer cells (Figures 10-11), and BB5, primary human breast cancer cells (Figures 12-13). For all cell types tested it was possible to encapsulate the cells with a hydrogel (PEG based or collagen) that did not reduce cell viability compared to organoids grown without encapsulation.

[0205] Methods / Results: Organoids were generated as described in Example 1 using MCF10A cells (Figure 6A) or OVCAR8 cells (Figure 6B). In brief, about 50 pL suspension of about 4000 MCF A or OVCAR8 cells was added to each well of a non-adherent imaging plate, formed into a spheroid with the magnetic device, and stained with TMRE (about 100nM, yellow) and MTC (about 1 uM, red). OVSRI-II cells were also stained with Annexin V-FITC (about 350ng / ml_, green), to visualize apoptotic cells as described in Example 1. For spheroids derived from cell line OVCAR8 cells, the indicated gel (PCT-Gel3 or Collagen) was added on Day 0 and the spheroids were imaged by confocal microscopy on day 7 with a 5X air lens. The process is benign as shown in the yellow staining indicative of metabolically active cells and the paucity of red staining indicative of stressed cells. To examine longer incubation times, patient derived OVSRI-II cells were formed into spheroids with the dyes (TMRE (100nM, yellow), Annexin V-FITC (350ng / ml_, green), MTC (1 uM, red) using a magnetic device and embedded in PCT-Gel1 on day 0 and the dyes were included in the media during all subsequent media changes. Viability was determined by visualizing the increase in size of the organoids and the number of TMRE positive cells (alive) in maximum intensity projections of confocal images recorded on the Phenix Opera automated spinning disc confocal microscope with a 20X air lens at days 7, 22 and 35 (Figure 10) and day 41 (Figure 11).

[0206] Results: As shown in Figure 6A for MCF 10A cells the main organoid is primarily TMRE positive (metabolically active) and not green (Annexin V negative, alive). Some cells that were not incorporated into, or exited from the structure, ended up on the bottom of the well where they died (green cells, apoptosis; blue nuclei without green or yellow cell bodies, died by an unknown mechanism). As shown in Figure 6B, after about 7 days in culture, the encapsulated organoids are predominantly yellow indicating that most of the hydrogel embedded OVCAR8 cells are metabolically active. The small amount of red staining indicates there are some (relatively few) stressed cells. As shown in Figures 10 and 11 , OVSRI-II patient derived organoids embedded in PCT-Gel1 continue to grow larger for about 41 days. The outer layer cells are stressed (red) with few dying cells (green) while the interior is predominantly yellow (metabolically active). Individual energized mitochondria are well resolved in confocal micrographs recorded using a 40X water immersion lens (Figure 11 B) confirming that the interior of the organoids remains viable.

[0207] As shown in Figure 11 A, Hoescht 33342 could not be added at Day 0 due to toxicity of the dye. In addition, at day 41 it was evident that the Hoechst (about 1 pg / ml, blue), that should stain nuclei, did not penetrate into the organoid very well even when added about 24 hours prior to imaging. In the left image some cells have stained nuclei (blue) but in the right image lots of the nuclei are dark because the dye did not penetrate the encapsulated organoid and the nucleus is not stained by the other dyes. The other dyes are non-toxic and therefore the organoid was grown in the presence of those dyes ensuring equivalent access throughout. For that reason, all the cells are evenly stained with the concentrations of TMRE, MTC and the annexin described in Example 1 (although there are not many green cells because the organoid is still growing).

[0208] Example 9 Comparison of non-adherent flat bottom coated plates vs agarose cups for growth and imaging of encapsulated primary human breast cancer organoids

[0209] The two different methods of generating encapsulated spheroids, magnetic aggregation and growth in non-adherent agarose cups, were compared for supporting the growth and imaging of primary human breast cancer cells. The exemplary cell culture, BB5, consists of a mixed population of cells obtained from a patient biopsy sample that was conditionally reprogrammed to enable growth of organoids.

[0210] Methods: Spheroids were generated, using BB5 cells, in polyHEMA coated flat bottom plates (Figure 13) as described in Example 6. Spheroids were also generated, using BB5 cells, incubated in agarose cups (Figure 14). Each of these spheroids were encapsulated in PCT-Gel4, a PEG based gel that has a crosslinking peptide that is degradable by matrix metalloproteases. The media included the dyes TMRE (yellow), MTC3 (red), AnnV (green), as described above. For spheroids generated using the magnetic device, encapsulation was on Day 0 immediately after the about 3-hour incubation required to generate spheroids. Spheroids generated using the agarose cups were encapsulated in PCT-Gel4 after overnight incubation. The spheroids reorganize into organoids during continued incubation and were imaged by confocal microscopy at day 27 (Figure 15) or day 14 (Figure 16). Hoescht dye (about 1 pig / ml), was added the day before imaging and the organoids were returned to the incubator. (A panels) Micrographs are maximum intensity projections of organoids located using PreciScan at 5x followed by imaging at 20x with an air lens. (B panels) Micrographs were maximum intensity projections of organoids located using PreciScan at 5x followed by confocal imaging with a 40x water immersion lens.

[0211] Results: Figure 12A-B, which differ only by magnification, show that PCT-Gel 4, like PCT- Gel 1 , can be used to encapsulate organoids. This typically involved a low viscosity (similar to water about 1 cP and less than about 10 cP) and gelling time (about 10 minutes). As shown in Figure 13A-B, encapsulating the organoids formed in agarose cups with PCT-Gel 4 can also keep them in place. The results of Figures 12 and 13 show that BB5 derived organoids exhibited less stress (less red) and more metabolic activity (yellow) at day 27 when grown in the polyHEMA coated plates in PCT-Gel4 than at day 14 when grown in agarose cups with collagen gel. It is not known if this reflects differences in the polyHEMA coated plates and agarose cups or is primarily due to imaging on different days. Nevertheless, organoids grew successfully in both conditions for at least 14 days.

[0212] Furthermore, there is not a significant difference is the image quality between the polyHEMA coated plates or the agarose cup. In addition, while not shown, other cell types, like prostate cancer cells, grew well in the tested conditions (data not shown). It is noted that agarose wells may require more calculation in the PreciScan sequence since a slight change in agarose deposition or organoid position can result in a relatively large difference in z position. As such, the flat bottom wells (with gel encapsulation) can allow for a faster collection of data.

[0213] Example 10 Organoids growth with The Well Bioscience alginate gel

[0214] The purpose of this study was to: 1) test 4 Vitrogels purchased from The Well Bioscience with several cell line organoids and monitor organoid growth; and 2) determine whether Vitrogels can be a potential substitution for other hydrogels;

[0215] Methods: Cell lines tested:

[0216] Organoid generation: The organoids were generated using two different methods:

[0217] (1) By using hydrogel cups described and exemplified herein: each well in the 384 well plate was coated with a hydrogel cup so that cells would gather at the bottom of the cup, thus reorganizing themselves into spheroids.

[0218] In this method, cells were seeded at about 500 cells / well at about 40pL and the plate was left overnight in about 37°C for spheroid formation. Next day, about 30pL of media was carefully removed from each well. Then, about 20pL of Vitrogel was added into each well. The plate was incubated at room temperature for about 15 mins for soft gel formation. Fresh media was added on top afterwards to bring the total volume to about 80pL. The plate was put back to the 37°C incubator. Media was exchanged every 2 to 3 days.

[0219] The plate layout was as follows:

[0220] Each color represented a cell line. Each gel was added horizontally in a row (i.e Gel 1 was added from C3 to C7 for Ovcar3). Each condition had 5 technical replicates to account for potential pipetting and imaging errors.

[0221] (2) By using a magnetic device: cells were forced together by magnetic field to become spheroids.

[0222] In this method, cells were seeded at about 500 cells / well at about 10pL in the paramagnetic salt solution. After about 4 hours incubation on the magnetic device in about 37°C, about 20pL of gel was added on top. The plate was incubated at room temperature for about 15 mins for soft gel formation. Fresh media was added slowly in each well afterwards to bring the total volume to about 80pL. The plate was removed from the magnetic device and was incubated at about 37°C for about 30 minutes. The media was exchanged twice to remove the salts. The media was exchanged again the next day. Thereafter, the media was exchanged every 2 to 3 days.

[0223] The plate layout was as follows:

[0224] Each color represented a cell line. Each gel was added horizontally in a row (i.e Gel 1 was added from 03 to C7 for Ovcar3). Each condition had 5 technical replicates to account for potential pipetting and imaging errors.

[0225] The spheroids were generated prior to adding the Vitrogels. The amount of gel used for each organoid is at 2:1 v / v mixing ratio as recommended (about 10pL cells + about 20pL gel). After the hydrogel was added the spheroids spontaneously reorganize in some hydrogels.

[0226] Results: Figures 14 to 20 illustrate the growth of different cell lines as spheroids using the magnetic aggregation and hydrogel cup methods. As shown in Figures 14-15 Ovcar3 cells form non-spherical structures with the magnetic (Figure 14) and hydrogel cup methods (Figure 15). Furthermore, in all 4 Vitrogels the Ovcar3 cells were mostly dying as indicated by low ChromaLive Yellow and bright AnnexinV signal. There is also evidence of nuclear swelling and chromatin condensation (Hoescht staining) in the Vitrogels. The Ovcar3 spheroids formed by either method survived better in the control (PCT) gel condition (brighter ChromaLive Yellow and lower AnnexinV signal compared to Vitrogels. There is some evidence of chromatin condensation but not nuclear swelling; see Figure 14-15).

[0227] Figures 16-17 illustrate that Ovcar8 spheroids were able to grow with all 4 Vitrogels and the control gel. However, the cells were very stressed (indicated by bright ChromaLive Red signal; see, Figure 16-17). Furthermore, the 3D structures formed differed depending on the aggregation method and the gel type. Magnetic aggregation resulted in relatively spherical structures however in Vitrogel 2 the center of the spheroid contained few viable cells as shown by the lower intensity of Hoescht stain. Vitrogel 1 and 3 resulted in spheroids composed of fewer cells as shown by Hoescht stain. In the hydrogel cups Ovcar8 cells grew in a more disorganized fashion while spherical structures were obtained using the Vitrogels. Additionally, there was a more even distribution of Hoescht stain in cup method with the Vitrogels.

[0228] Figures 18-19 illustrate that when aggregated using the magnet device the 4 vitrogels resulted in larger LnCap spheroids than the control gel. The spheroids formed in Vitrogels 1 and 4 were hollowing out more clearly when formed in the magnetic device compared to hydrogel cups. Spheroids formed with the magnetic device exhibited more chromatin condensation in Vitrogel 2 than in the other gels. When formed in the hydrogel cups LnCap cells formed spheroids formed in all 4 vitrogels and the control gel were similar in size and intensities of the dyes (see, Figures 18-19).

[0229] Figures 20-21 illustrate that PC3 cells formed viable spheroids only in the hydrogel cup with the control gel. In Vitrogels the PC3 cells did not form spheroids with either method however, the cells remained viable (low AnnexinV staining and limited chromatin condensation) but showed evidence of stress (high CromaLive Red intensity). The variable ChromaLive yellow intensity indicates some reduced metabolism, (see, Figure 20-21).

[0230] It was additionally found that, in both organoid generation methods, after soft gel formation, when adding more media to each well to cover the Vitrogel, the organoids moved around in the well. The control gel was able to keep the organoids in place.

[0231] Example 11 Primary mesenteric metastasis of colon cancer and small bowel cancer patient derived organoids

[0232] In this Example, organoids were made with the hydrogel cup method described and exemplified herein and stained with Chromalive stain plus Hoescht stain. Figures 22-24 show a Z-stack of confocal images of a single organoid from a mesenteric metastasis of colon cancer. Optical sections begin below the organoid and are of increasing height through the organoid. The object was imaged using a 20X water immersion lens and Opera Phenix automated spinning disk confocal microscope. The results of this Example are shown as Figures 22 to 26.

[0233] Figure 22 shows the location of nuclei and therefore cells in the Z-stack of images as indicated by Hoescht staining. As Z increases there are fewer nuclei in the center of the structure indicating that there are no cells in the center of the organoid. There is also an opening in the structure suggesting formation of a villus type structure.

[0234] Figure 23 shows metabolic activity as shown by ChromaLive yellow staining of the same optical sections shown in Figure 22. Yellow staining in the area associated with nuclei indicates viable metabolizing cells surround the center of the structure and opening that are devoid of cells.

[0235] Figure 24 shows that the areas of the organoid devoid of cells are filled with material that stains with ChromaLive red.

[0236] Figure 25 shows an enlargement of 4 of the confocal sections of the same organoid and the interpretation of the ChromaLive red staining patterns. ChromaLive red staining in the nucleus indicates a dead cell. Stressed cells have higher intensity ChromaLive red staining in the cytoplasm than normal cells. Chromalive Red stained debris has a different texture than seen in cells.

[0237] Figure 26 illustrates staining by Chromalive and Hoescht overlaid on the same optical sections of the organoid to enable colocalization of the different colors to be visualized and highlight villus formation.

[0238] In summary, the hydrogel cup method can be used to form organoids that when stained with Chromalive and Hoescht, can allow for one to obtain high quality spatial information in primary patient derived organoids derived from a variety of different tissues.

[0239] Example 12 Optimizing conditions for 3D cell culture using hydrogel cups and different surrounding matrices

[0240] Methods / Results: Hydrogel cups were formed using about 900nl of about 1% Hydrogel per well of a 1536 well plate (Plate - Cell Carrier Square). About 1000 cells per well were seeded into hydrogel cups on day 0. 2 marginal columns and two marginal rows of the plate were not imaged as they were filled with PBS and were not used in the experiment to avoid edge effects. After overnight incubation to form spheroids in the wells, the indicated matrix was added (approximate final concentrations as indicated). MD is Collagen I about 500 pg / ml; iMatrix about 50 pg / ml; and Fibronectin about 50 pg / ml; and MC is Collagen I about 1000 pg / ml; iMatrix about 100 pg / ml; and Fibronectin about 100 pg / ml. The results of this Example are shown as Figures 27-31 . Organoids were imaged in bright field and by confocal imaging of fluorescence using an Opera Phenix automated spinning disk confocal microscope. Organoids were stained with Hoescht to visualize nuclei (blue), TMRE (yellow) to indicate mitochondrial inner membrane transmembrane potential and Annexin (green) to indicate apoptotic cells or cell masses / debris. Figure 27 shows formation of primary human breast adenoma and cancer organoids with hydrogel cups described and exemplified herein. 3D cultures in hydrogel cups can be surrounded by various materials serving as artificial extracellular matrix. 3D cultures were successfully embedded and grew in iMatrix, low concentrations of collagen, fibronectin, MD and MC matrices. At higher concentrations of collagen (>100 ug / ml) the density of the matrix resulted in the 3D cultures rising above the bottom and moving out of the center of the wells. Primary cells from two patients are shown (BB1 and BB5#1). BB1 is derived from a breast adenoma and by day three organoids grew much larger in all of the matrices than in the absence of matrix (matrix concentration 0). BB5#1 were derived from a breast carcinoma and at day 3 were approximately the same with and without added matrix. “Fluor beads” indicates hydrogel cups containing fluorescent beads (not visible) used to measure the cup dimensions using the method shown in Figure 2. The imaging plane was about 120 pM above the bottom of the well. Bright field images were acquired using a 10x air objective.

[0241] Figure 28 shows formation of primary human breast cancer and adenoma organoids with hydrogel cups and 1000 pg / ml collagen described and exemplified herein. These organoids have floated in the collagen to the heights indicated (815-1160 pm above the bottom of the wells). Nevertheless, organoid formation was successful as indicated by the images.

[0242] Figure 29 shows confocal optical slices of primary human breast cancer organoids of Example 12 at Day 3; collagen I 100 pg / ml; 40x magnification, water immersion lens.

[0243] Figure 30 shows confocal optical slices of primary human breast cancer organoids of Example 12 at Day 3; Fibronectin 100 pg / ml; 40x magnification, water immersion lens.

[0244] Figure 31 shows confocal optical slices of primary human breast cancer organoids of Example 12 at Day 3; iMatrix 50 pg / ml; 40x magnification, water immersion lens.

[0245] The above disclosure generally describes the present invention. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.

[0246] All publications, patents and patent applications cited above are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

[0247] Although preferred embodiments of the invention have been described herein in detail, it will be understood by those skilled in the art that variations may be made thereto without departing from the spirit of the invention or the scope of the appended claims.

Claims

We claim:1 . A gellable material for encapsulating a cellular aggregate in a well plate.

2. The gellable material of claim 1 , wherein the cellular aggregate, once encapsulated, has substantially no movement in the well plate during media exchange, handling of the well plate or tilting of the well plate.

3. The gellable material of claim 1 , wherein the cellular aggregate, once encapsulated, has no movement in the well plate during media exchange, handling of the well plate or tilting of the well plate.

4. The gellable material of any one of claims 1 to 3, wherein the cellular aggregate, once encapsulated, stays in a position in the well plate.

5. The gellable material of claim 4, wherein the position is relative to the x, y, and z axis of an imaging microscope.

6. The gellable material of claim 4 or 5, wherein the position is at a bottom of the well plate.

7. The gellable material of any one of claims 1 to 6, wherein the gellable material is selected from collagen, fibronectin, Vitrogel™, iMatrix™, MD, or MC.

8. The gellable material of any one of claims 1 to 6, comprising a non-protein hydrogel.

9. The gellable material of claim 8, wherein the non-protein hydrogel comprises polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), polyglycolic acid (PGA), polyvinyl alcohol (PVA), hyaluronan (HA) gels, alginate gels, or fibrin gels.

10. The gellable material of claim 9, wherein the non-protein hydrogel comprises PEG.

11. The gellable material of claim 9 or 10, wherein the PEG is a modified PEG.

12. The gellable material of claim 11 , wherein the modified PEG comprises a PEG crosslinked by a peptide.

13. The gellable material of any one of claims 1 to 6, comprising a protein hydrogel.

14. The gellable material of claim 13, wherein the protein hydrogel comprises collagen, elastin, gelatin, fibrin, fibronectin or combination thereof.

15. The gellable material of claim 13 or 14, wherein the protein hydrogel comprises collagen.

16. The gellable material of any one of claims 1 to 15, wherein the gellable material is degradable.

17. The gellable material of any one of claims 1 to 16, wherein the gellable material has a low viscosity.

18. The gellable material of any one of claims 1 to 17, wherein the viscosity is about 0.5 mPa.s to about 10 mPa.s.

19. The gellable material of any one of claims 1 to 16, wherein the gellable material is not viscous.

20. The gellable material of any one of claims 1 to 19, wherein the gellable material has a convenient gelling time to encapsulate the cellular aggregate.21 . The gellable material of any one of claims 1 to 20, wherein the gelling time is between about 1 minute to about 180 minutes.

22. The gellable material of claim 21 , wherein the gelling time is about 10 minutes, 30 minutes, or 60 minutes.

23. The gellable material of any one of claims 1 to 22, wherein the well plate is a flat bottom well plate.

24. The gellable material of any one of claims 1 to 22, wherein the well plate is a rounded bottom well plate.

25. The gellable material of any one of claims 1 to 24, wherein the well plate is made non-adherent with a non-adherent coating.

26. The gellable material of claim 25, wherein the non-adherent coating comprises a covalently bound hydrogel layer selected from Corning™ ultralow attachment range, Thermo Fisher™ Nunclon Sephera range, or polyHEMA (poly-2-hdroxylethyl methacrylate).

27. The gellable material of any one of claims 1 to 26, wherein the cellular aggregate, before or after encapsulation, can mature into an organoid or spheroid.

28. The gellable material of any one of claims 1 to 27, wherein the cellular aggregate, before or after encapsulation, can mature into an organoid or spheroid for up to about 50 days.

29. The gellable material of any one of claims 1 to 28, wherein the cellular aggregate, before or after encapsulation, is an organoid or a spheroid.

30. The gellable material of claim 29, wherein the organoid or spheroid is produced from cells derived from various tissues or organs, such as skin, breast, lung, liver, pancreas, kidney, blood, stomach, intestine, spleen, ovary, heart, cervix, bladder, oral cavity, esophagus, prostate, or mesentery.31 . The gellable material of any one of claims 27 to 30, wherein the organoid or spheroid is derived from stem cells or tumor cells such as a primary tumor cell or a tumor cell line.

32. The gellable material of any one of claims 1 to 31 , wherein, after encapsulation, the cellular aggregate is suitable for imaging or screening.

33. A method for producing an encapsulated cellular aggregate in a well plate, the method comprising: adding a gellable material to a cellular aggregate in the well plate; andforming the encapsulated cellular aggregate by gelling the gellable material.

34. The method of claim 33, wherein the adding comprises diffusing the gellable material over a period of time.

35. The method of claim 34, wherein the diffusing comprises adding the gellable material to the well plate gently so as to not damage the cellular aggregate.

36. The method of claim 34 or 35, wherein the diffusing comprises layering the gellable material so that the gellable material can mix with a culture media around the cellular aggregate in the well plate.

37. The method of any one of claims 34 to 36, wherein the period of time is about 1 minute to about 180 minutes.

38. The method of any one of claims 33 to 37, wherein the gelling causes the encapsulated cellular aggregate to stay in a position in the well plate.

39. The method of any one of claims 33 to 38, further comprising growing cells into the cellular aggregate in a culture medium prior to the adding the gellable material.

40. The method of any one of claims 33 to 39, wherein the gelling of the gellable material occurs for a period of time.41 . The method of claim 40, wherein the period of time is about 1 minute to about 180 minutes.

42. The method of claim 40, wherein the gelling time is about 10 minutes, 30 minutes, or 60 minutes.

43. The method of any one of claims 33 to 42, further comprising adding a dye compound to the well plate prior to the adding.

44. The method of any one of claims 33 to 42, further comprising adding a dye compound to the well plate after the forming the encapsulated cellular aggregate.

45. The method of claim 43 or 44, wherein the dye compound is selected from:5, or a salt thereof.

46. The method of any one of claims 43 to 45, wherein the dye compound allows for detecting or monitoring changes in cell state (e.g., cell physiology, behavior) in response to an agent or stimuli such as changes in pH, temperature, salt concentration, contact with other cells, or treatment with a drug or drug candidate.

47. The method of any one of claims 33 to 46, further comprising adding a drug, such as an anti-cancer drug, to the encapsulated cellular aggregate.

48. The method of any one of claims 33 to 47, further comprising growing cells, such as fibroblasts, on the encapsulated cellular aggregate.

49. The method of any one of claims 33 to 48, further comprising maturing the cellular aggregate, before or after encapsulation, into spheroids over a period of time.

50. The method of any one of claims 33 to 49, further comprising maturing the cellular aggregate, before or after encapsulation, into organoids over a period of time.51 . The method of claim 50, wherein the period of time is about 50 days.

52. The method of any one of claims 33 to 51 , wherein the cellular aggregate, before or after encapsulation, is a spheroid or an organoid.

53. The method of claim 52, wherein the organoid or spheroid is produced from cells derived from various tissues or organs, such as skin, breast, lung, liver, pancreas, kidney,blood, stomach, intestine, spleen, ovary, heart, cervix, bladder, oral cavity, esophagus, prostate, or mesentery.

54. The method of any one of claims 49 to 53, wherein the organoid or spheroid is derived from stem cells or tumor cells such as a primary tumor cell or a tumor cell line.

55. The method of any one of claims 33 to 54, further comprising adding a paramagnetic agent to the well plate prior to the adding.

56. The method of claim 55, wherein the paramagnetic agent comprises a Gadolinium based salt or contrasting agent, optionally Gadolinium-diethylenetriamine penta-acetic acid (Gd-DTPA).

57. The method of any one of claims 33 to 56, wherein the well plate is a flat bottom well plate.

58. The method claim 57, wherein the flat bottom well plate comprises a non-adherent coating selected from Corning™ ultralow attachment range, Thermo Fisher™ Nunclon Sephera range, or polyHEMA (poly-2-hdroxylethyl methacrylate).

59. The method of any one of claims 33 to 56, wherein the well plate is a rounded bottom well plate.

60. The method of any one of claims 33 to 59, wherein, after encapsulation, the cellular aggregate is suitable for imaging or screening.61 . The method of any one of claims 33 to 60, wherein the gellable material is selected from collagen, fibronectin, Vitrogel™,iMatrix™, MD, or MC.

62. A method of forming a non-adherent hydrogel cup for cellular aggregate formation, dispensing a non-adherent hydrogel to a well plate at a temperature, wherein the non-adherent hydrogel is sufficient viscous to form the non-adherent hydrogel cup having a curved surface.

63. The method of claim 62, further comprising allowing the non-adherent hydrogel to cool.

64. The method of claim 62 or 63, wherein the temperature is about 45°C to about 60°C.

65. The method of any one of claims 62 to 62, wherein the higher the viscosity of the non-adherent hydrogel, the steeper the sides of the cup formed.

66. The method of any one of claims 62 to 63, wherein well plate has a flat imaging bottom and round wells.

67. The method of any one of claims 62 to 63, wherein the well plate does not have square wells.

68. The method of any one of claims 62 to 67, wherein a shape of the non-adherent hydrogel cup is determined by the viscosity of the non-adherent hydrogel while in liquid form.

69. The method of any one of claims 62 to 68, wherein the non-adherent hydrogel cup can support growth / differentiation of cells into the cellular aggregate.

70. The method of any one of claims 62 to 69, wherein the non-adherent hydrogel has a refractive index close to that of the cells of the cellular aggregate.71 . The method of any one of claims 62 to 70, wherein the non-adherent hydrogel is agarose.

72. The method of any one of claims 62 to 71 , wherein the cellular aggregate can be encapsulated by the gellable material of any one of claims 1 to 32.

73. The method of any one of claims 62 to 72, wherein the organoid or spheroid is produced from cells derived from various tissues or organs, such as skin, breast, lung, liver, pancreas, kidney, blood, stomach, intestine, spleen, ovary, heart, cervix, bladder, oral cavity, esophagus, prostate, or mesentery.

74. The method of any one of claims 62 to 72, wherein the organoid or spheroid is derived from stem cells or tumor cells such as a primary tumor cell or a tumor cell line.

75. Use of the gellable material of any one of claims 1 to 32 for encapsulating a cellular aggregate.

76. The use of claim 75, wherein, after encapsulation, the cellular aggregate is suitable for imaging or screening.

77. The use of claim 75 or 76, wherein the cellular aggregate, before or after encapsulation, is an organoid or a spheroid.